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Stem Cells, Vol. 19, No. 3, 236-246, May 2001
© 2001 AlphaMed Press

Hybrid HIV/MSCV LTR Enhances Transgene Expression of Lentiviral Vectors in Human CD34+ Hematopoietic Cells

John Kim Choia, Nghia Hoanga, Antonina M. Vilardia, Patricia Conradc, Stephen G. Emersonb, Alan M. Gewirtzb

a Department of Pathology and Laboratory Medicine and
b Department of Medicine, University of Pennsylvania, Philadelphia, USA;
c Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA

Key Words. Lentiviral vector • MSCV-based vector • Human CD34+ cells • Hybrid LTR

John Kim Choi, M.D., Ph.D., University of Pennsylvania, 413A SCL, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104, USA. Telephone: 215-573-6527; Fax: 215-573-6523; e-mail: jkchoi{at}mail.med.upenn.edu;  Alan M. Gewirtz, M.D., University of Pennsylvania School of Medicine, Department of Pathology and Internal Medicine, Rm 713 BRB II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104, USA. Telephone: 215-898-4499; Fax: 215-573-2078; e-mail: gewirtz{at}mail.med.upenn.edu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Viral Concentration Increases...
 Discussion
 References
 
HIV-based lentiviral vectors can transduce nondividing cells, an important advantage over murine leukemia virus (MLV)-based vectors when transducing slowly dividing hematopoietic stem cells. However, we find that in human CD34+ hematopoietic cells, the HIV-based vectors with an internal cytomegalovirus (CMV) promoter express transgenes 100- to 1,000-fold less than the MLV-based retroviral vector murine stem cell virus (MSCV). To increase the expression of the integrated lentivirus, we replaced CMV promoter with that of the Rous sarcoma virus or MSCV and obtained a modest augmentation in expression. A more dramatic effect was seen when the CMV enhancer/promoter was removed and the HIV long-terminal repeat (LTR) was replaced by a novel HIV/MSCV hybrid LTR. This vector retains the ability to transduce nondividing cells but now expresses its transgene (enhanced green fluorescent protein) 10- to 100-fold greater than the original HIV-based vector. When compared under identical conditions, the HIV vector with the hybrid LTR transduced a higher percentage of CD34+ cells than the MSCV-based retroviral vector (19.4% versus 2.4%). The number of transduced cells and level of transgene expression remain constant over 5-8 weeks as determined by long-term culture-initiating cells, fluoresence-activated cell sorting, and nonobese diabetic/severe combined immunodeficiency repopulation assay.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Viral Concentration Increases...
 Discussion
 References
 
HIV-based vectors are attractive vehicles for delivering transgenes into slow to nondividing cells such as human hematopoietic stem cells [1-5], neurons [6], skeletal myocytes [7], and hepatocytes [7]. However, one significant disadvantage of the HIV-based vectors for hematopoietic applications is the weak activity of the HIV long-terminal repeat (LTR) in cells of this lineage. To increase the activity, HIV-based vectors that express the HIV accessory protein tat have been engineered [1, 3, 5]. A concern with this strategy is that the tat protein enhances transcription of other cellular genes that may be detrimental to the cells [8-13]. Furthermore, tat protein may also enter into neighboring cells [14] with similar effects. Other first and second generation HIV-based vectors do not express tat but use an internal cytomegalovirus (CMV) promoter to drive the expression of the transgene [2, 4].

While exploring various experimental systems to express ectopic oncogenes in primary human CD34+ cells, we found that the HIV-based vectors with an internal CMV poorly express its transgene when compared to the murine leukemia virus (MLV)-based vector murine stem cell virus (MSCV). This poor expression could hamper studies that require a threshold quantity of transgene for a biologic effect. The poor expression also has clinical implications in gene therapy. To explore approaches for increasing transgene expression, we engineered various modifications of a first generation HIV-based vector driven by an internal CMV enhancer/promoter [15]. Of these, transgene expression was highest with a vector in which the internal CMV enhancer/promoter was deleted and a portion of the U3 region of the 3' HIV LTR was replaced with the U3 region of MSCV LTR. The 3' hybrid LTR duplicates and replaces the 5' LTR during viral reverse transcription leading to expression that is driven by the HIV/MSCV hybrid enhancer/promoter. This modified vector retains the property to transduce nondividing cells and expresses its transgene 10-100-fold greater than the original HIV-based vector containing the internal CMV enhancer/promoter.


   MATERIALS AND METHODS
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 Abstract
 Introduction
 Materials and Methods
Results
 Viral Concentration Increases...
 Discussion
 References
 
Vectors
The original HIV vector pHR'CMVLacZ, the packaging plasmid CMV{Delta}R8.2, and the VSV-G envelope plasmid pMD.G [6] were generously provided by Inder M. Verma, Salk Institute; La Jolla, CA. The MSCV-based vector MIGR1 [16] was generously provided by Warren Pear, University of Pennsylvania, Philadelphia, PA. The RSV and MSCV enhancer/promoters were isolated by polymerase chain reaction (PCR) amplification of the pBK-RSV (Stratagene; La Jolla, CA; http://www.Stratagene.com) and MIGR1 plasmids, respectively. The hybrid MSCV/HIV LTR was generated by PCR amplification and extension using the primers listed in Table 1Go. The PCR products were verified by sequencing and introduced into the original HIV vector using standard methods.


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  		Table 1. Oligonucleotides to generate enhancer promoter
 
Generation of Virus

MSCV-Based Retrovirus   Retroviruses were generated using previously described methods [16]. Briefly, MIGR1 and a vector that encodes the viral envelope protein VSV-G were transiently cotransfected into the packaging cell line glycoprotein (GP) (generously provided by Garry P. Nolan, Stanford University Medical Center; Stanford, CA). During the next 3 days, the transfected GP cells release retroviruses into the culture medium; each ml of media contains 105 to 107 infectious viral particles using K562 as target cells. The culture media were collected 48 and 72 hours post-transfection and stored in small aliquots at -70°C.

HIV-Based Lentivirus   Lentiviral particles were generated in 293T cells using transient transfection with plasmids encoding the RNA genome, viral protein, and VSV-G envelope protein following described methods [6]. Viral particles were collected and stored as described for the MSCV-based retrovirus. Viral collection was tested for replication-competent viruses by detecting for rescue of integrated provirus that expresses enhanced green fluorescent protein (EGFP).

Viral Concentration   VSV-G pseudotyped retroviruses were concentrated by filtration using Biomax low-speed centrifuge filtration concentration (Millipore; Medford, MA).

The titer of the virus was determined by transducing K562 as target cells, and the number of EGFP+ cells was determined by fluorescence-activated cell sorter analysis (FACS) 5-7 days later.

Transduction
Four-hundred thousand primary CD34+ cells, 50 µl of unconcentrated or concentrated virus, and 8 µg/ml of polybrene (Sigma; St. Louis, MO; http://www.sigma-aldrich.com) were centrifuged in a 96-well plate at 1,500 x g for 90 minutes at room temperature following published protocols [17]. For all experiments, cells were transduced using a single exposure to the viral stock. Afterwards, the cells were washed once with 10 volumes of serum-free media and then cultured.

Tissue Culture of Cells

Cell Lines   K562 cells were cultured in RPMI supplemented with 10% fetal bovine serum (FBS). 293T, GP, and HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS. Nondividing HeLa cells were established using published protocols [18]. Briefly, HeLa cells were seeded to generate plates that were 25% confluent and then treated with aphidicolin (15 µg/ml) 24 hours prior to transduction and daily thereafter.

Primary Human Hematopoietic Cells   Cells from bone marrow (BM) aspirates or umbilical cord blood (UCB) were purified by Ficoll gradient and plated in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% FBS at 5-10 million cells/ml. After overnight incubation, the nonadherent mononuclear cells were further processed for CD34+ cells by positive immunomagnetic selection using the MACS system (Miltenyi Biotec; Auburn, CA; http://www.miltenyibiotec.com). CD34+ cells were cultured in IMDM supplemented with 10.0% FBS supplemented with the growth factors stem cell factor (SCF, 100 ng/ml), thrombopoietin (TPO, 50 ng/ml), and flk-3 (100 ng/ml) and the cells cultured for 2-3 days prior to transduction. For transduction without growth factors, freshly isolated CD34+ cells were transduced.

Long-Term Bone Marrow Culture   Primary CD34+ BM or UCB cells were transduced and cultured on a monolayer of irradiated primary BM stromal cells for 5-8 weeks with IMDM supplemented with 12.5% horse serum and 12.5% FBS following published protocols [19]. At weekly intervals, half of the media was replaced with fresh media and cells were demi-depleted. After 5-8 weeks, unattached cells and trypsinized attached cells were cultured in methylcellulose (Stem Cell Technology; Vancouver, BC, Canada; http://www.stemcell.com) supplemented with SCF (100 ng/ml), interleukin 3 (IL-3, 20 ng/ml), IL-6 (20 ng/ml), IL-11 (50 ng/ml), GM-CSF (20 ng/ml), and erythropoietin (1 µ/ml). Colonies were scored 10-15 days later under phase and ultraviolet (UV) microscopy.

Animals
A colony of NOD/LtSZ-scid/scid mice (NOD/SCID) was established in the animal facility of the University of Pennsylvania. They were housed in microisolator cages and provided with autoclaved food and water. For the repopulation assay, mice were irradiated with two 150 cGy fractions 2 hours prior to injection. 2 x 106 transduced UCB cells were injected via tail vein. After 5 weeks, the mice were sacrificed, and BM cells were isolated from the femurs and tibias.

DNA Isolation
Genomic DNA was isolated using the QIAamp DNA purification columns (Qiagen; Valencia, CA; http://www.qiagen.com.) using manufacturer's protocol. DNA was quantified by UV absorption spectrophotometry and ethidium bromide binding.

PCR
PCR was performed using standard procedure. Briefly, 5 ng of genomic DNA was amplified in 50 µl reaction for 30 cycles using 0.5 units of Amplitaq Gold (Perkin Elmer; Norwalk, CT; http://www.instruments.perkinelmer.com/index.asp), 0.5 uM of each primer, and PCR parameters that consisted of denaturing at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and elongating at 72°C for 30 seconds. EGFP primer sequences were 5' ATC CTG GTC GAG CTG GAC GGC and 5' CGT GCT GCT TCA TGT GGT CGG G. Insulin receptor primer sequences were 5' GGA ACA ACC TCA CTA GGT TGC A and 5' CTG GCA GTG ACT ATG AGT CCA A.

FACS Analysis
FACS analysis was performed on FACSTAR and analyzed using Cell-Quest software (Becton Dickinson; San Jose, CA; http://www.bd.com). Phycoerythrin-conjugated antibodies to CD34 and CD45 were purchased from PharMingen (San Diego, CA; http://www.pharmingen.com).


   RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
Viral Concentration Increases...
 Discussion
 References
 
Integration and Expression of HIV Lentiviral Vector Compared to MSCV Vector
The original HIV-based lentiviral vector encodes for beta-galactosidase while the MSCV-based MIGR1 retroviral vector encodes for EGFP. The different transgenes in the two vectors require different detection systems and thus prevent direct comparison of the transduction efficiencies. To better compare the two vectors, the beta-galactosidase sequence of the original HIV vector was replaced with the internal ribosomal entry site (IRES) and EGFP from the MIGR1 vector (Fig. 1Go). The transgene EGFP allows rapid and easy determination of transduction using FACS analysis. The percentage of cells that express EGFP will correlate with the number of cells that have viral integration. Furthermore, the mean fluorescent intensity (MFI) of the EGFP in an individual cell will correlate with the level of expression of the integrated virus.



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  		Figure 1. Diagram of constructed retroviral vectors. The two parental vectors MIGR1 and pHR'CMVLacZ were obtained and modified using standard and PCR cloning techniques. To directly compare between MSCV and HIV-based systems, the multicloning site (MCS) and LacZ of the original lentiviral vector were replaced with the MCS, internal ribosomal entry site, and enhanced green fluorescent protein (EGFP) from a bicistronic MSCV-based vector. LTR = long terminal repeats; {psi} = viral packaging signal; CMV = CMV enhancer/promoter; RSV = RSV enhancer/promoter; MSCV = MSCV enhancer/promoter; MCS = multicloning site; IRES = internal ribosomal entry site; GFP = enhanced green fluorescent protein.

 
Lentiviral particles were generated in 293T cells using transient transfection with vesicular stromatitis virus glycoprotein (VSV-G) pseudotyping. MIGR1 viral particles were generated in GP cells using transient transfection with VSV-G pseudotyping. 105-107 infectious particles were routinely obtained per ml of harvested supernatant using K562 cells as the target cells. Following normalization of the titer, the viral supernatants were then used to transduce primary CD34+ cells that were isolated from adult BM aspirates or UCB and stimulated with SCF, TPO, and flk-3 for 2-3 days. Similar transduction efficiencies were obtained for 2 or 3 days of cytokine stimulation (data not shown). Six to 10 days post-transduction, the percentage of EGFP+ cells and their average level of EGFP expression were determined by FACS analysis. The MIGR1 retroviral vector expressed its EGFP 2-3 logs over control cells while the HIV lentiviral vector routinely expressed its EGFP only 0.25 to 0.5 logs over control cells (Fig. 2AGo). The significant overlap of the fluorescent pattern between the control and lentiviral-transduced cells prevented accurate determination of percentage of cells transduced. While the HIV lentiviral vector poorly expressed EGFP, the number of cells expressing EGFP appeared higher compared to the MSCV vector. This finding suggested that the lentiviral vector may have had a higher integration rate. To substantiate this impression, genomic DNA was isolated and the level of viral integration was semi-quantified by PCR for EGFP sequences (Fig. 2BGo). The number of cycles was empirically determined to be within the linear range (data not shown). In agreement with the FACS analysis, PCR confirmed the increased level of integration using the lentiviral vector compared to the MSCV based-vector. These findings suggest that the HIV-based vector integrated efficiently into cellular genome but expressed poorly when compared directly to the MSCV-based vector.



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  		Figure 2. Compared to MLV-based vector, HIV-based vector integrates better but expresses worse. A) BM CD34+ cells were isolated, transduced with either MIGR1 or pHR'CMVGFP, and analyzed 6 days later by FACS for EGFP fluorescence. B) Integration rate was also verified by isolating genomic DNA and detecting for EGFP by semi-quantitative PCR. A standard curve was generated by using control K562 genomic DNA spiked with varying percentages of genomic DNA from a stable EGFP+ K562 cell line. PCR for insulin receptor gene was done to confirm equal sample loading. Results are representative of three independent experiments.

 
Effect of Replacing the Internal CMV Enhancer/Promoter with Internal RSV or MSCV LTR on Transgene Expression in Human Hematopoietic Cells
The CMV enhancer/promoter expresses poorly in hematopoietic cells [20, 21]. We hypothesized that replacing the CMV enhancer/promoter with ones that express well in hematopoietic cells would generate more useful vectors. To test this hypothesis, we replaced the CMV enhancer/promoter with either the RSV enhancer/ promoter or the LTR enhancer/promoter of the MSCV-based retrovirus, two enhancer/promoters that can express well in hematopoietic cells [22-24]. These vectors, designated pHR'RSVGFP and pHR'MSCVGFP (Fig. 1Go) were transiently transfected with 293T cells with helper and VSV-G-encoding plasmids and the resulting viral supernatant used to transduce primary CD34+ cells. FACS analysis after 6-10 days demonstrated a modest increase (0.5-1 log over control fluorescence) in the expression of the transgene (Fig. 3Go).



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  		Figure 3. Replacing the internal CMV with RSV and MSCV promoter/enhancers increased the expression of the transgene in hematopoietic cells and expression is further increased by partial replacement of HIV LTR with the MSCV promoter. UCB CD34+ cells were isolated, transduced with either pHR'RSVGFP, pHR'MSCVGFP, or HIV/MSCV hybrid virus and analyzed 6 days later by FACS for EGFP fluorescence. MFI = mean fluorescent intensity. Results are representative of two independent experiments.

 
Effects of a Hybrid MSCV/HIV LTR on Lentiviral Transgene Expression in Human Hematopoietic Cells
Although the transgene expression was increased using an internal MSCV enhancer/promoter, the signal remained 2 logs weaker than that of the MSCV-based vector in which the transgene expression is driven off the LTR. We hypothesized that the MSCV enhancer/promoter may function better in the context of the LTR than as an internal element. To test this theory, we engineered another vector in which the MSCV LTR was placed not as an internal enhancer/promoter but as a partial replacement for the U3 region of HIV LTR that contains the HIV LTR enhancer/promoter. The ends of the U3 region of the HIV LTR were retained because the 5' end is one of the integration attachment sites [25] and the 3' end of U3 LTR is important for RNA processing [26]. To generate the hybrid LTR, 50 bp of the 5' end of the HIV U3 region, the complete U3 region of the MSCV LTR, and the 50 bp of the 3' end of the HIV U3 region with the complete HIV RU5 region were individually amplified by PCR (Fig. 1Go). The resulting three fragments were joined together by extension PCR and the resulting hybrid HIV/MSCV LTR replaced the HIV 3' LTR. During viral replication, the U3 region of the 3' LTR is duplicated and replaces the equivalent region in the 5' LTR [27]. The vector, helper and envelope plasmids were transiently transfected to generate virus. Viral collection was tested for replication-competent viruses by detecting for rescue of integrated provirus that expresses EGFP. No replication-competent viruses were detected.

CD34+ cells were transduced with the hybrid virus. Unconcentrated viral supernatant was used to reduce the possibility of multiple infection of a single cell and insure that the signal was produced from a single viral particle infection and transduction. FACS analysis after 6-10 days later demonstrated an increased expression (1.5-2 logs over control fluorescence) of the transgene compared to the cell transduced with the internal MSCV promoter (Fig. 3Go).

To verify that the hybrid virus can transduce CD34+ cells, transduced cells were stained with antibodies to CD34 and studied using two-color FACS analysis (Fig. 4AGo). Transduced CD34+ cells expressed their EGFP fluorescence nearly 2 logs over control and formed a distinct peak (Fig. 4AGo). The hybrid virus transduced 6%-8% (n = 3) of the total cells. Of the CD34+ cells, 6%-8% were EGFP+. Under our culture conditions, CD34 expression is slowly lost over 6-10 days such that approximately 10%-20% of the cells remain CD34+. The similar percentage of transduced cells in the CD34+ and CD34– populations suggests that the hybrid vector can transduce both populations equally well.



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  		Figure 4. Hybrid MSCV/HIV vector expresses well in hematopoietic progenitor cells. A) UCB CD34+ cells were isolated, transduced with the hybrid-based virus, and analyzed 6 days later by FACS for EGFP fluorescence and CD34 expression. B) Transduced UCB CD34+ cells were plated onto methylcellulose cultures with IL-3 and GM-CSF. A representative colony was photographed under phase and fluorescent microscopy. Results are representative of three or more independent experiments.

 
To verify that human progenitor cells are transduced by the hybrid LTR-based vector, colony-forming assays were performed. CD34+ cells were transduced and after 7 days, plated on methylcellulose tissue culture plates supplemented with the growth factors IL-3 and GM-CSF. Myeloid colonies expressing EGFP were evident under UV fluorescent microscopy (Fig. 4BGo). Approximately 5%-10% of the colonies were EGFP+, similar to the percentage seen by FACS analysis.


   VIRAL CONCENTRATION INCREASES TRANSDUCTION EFFICIENCY
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 Abstract
 Introduction
 Materials and Methods
 Results
 Viral Concentration Increases...
Discussion
 References
 
To increase the transduction rate, the viral supernatant was concentrated by using a low-speed filter concentration. Dilution and titering on K562 cells demonstrated 80%-90% recovery of viral transduction activity (data not shown). Twenty-fold concentrated virus increased the transduction rate of human CD34+ cells from 5%-9% to 10%-32% (Table 2). Higher concentration resulted in similar or reduced numbers.


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  		Table 2. Transduction efficiency: BM or UCB CD34+cells were isolated, transduced, and analyzed by FACS for EGFP fluorescence
 
Lentiviral Vector with Hybrid MSCV/HIV LTR Transduce Nondividing Cells
To determine if the hybrid vector retained the ability to transduce nondividing cells, HeLa cells were arrested in G1/S phase using the DNA polymerase inhibitor aphidicolin and then transduced with either the MSCV-based virus or the hybrid LTR lentivirus. After 3-4 days, the cells were trypsinized and analyzed by FACS for EGFP expression. The percentages of EGFP+ cells were normalized to those seen in dividing HeLa cells. The hybrid virus transduced greater than 60% of nondividing cells while the MSCV-based virus transduced less than 1% (data not shown).

The original HIV-based vectors can transduce CD34+ cells in the absence of exogenous growth factors [1, 2, 4]. To determine if the hybrid lentiviral vector retained this property, CD34+ cells were transduced with the concentrated hybrid virus in the absence of the exogenous growth factors SCF, TPO, flk-3. Under these conditions, the hybrid virus still transduced the cells although the efficiency decreased (Table 2, Fig. 5Go). However, the absence of growth factors had no significant effect on the level of EGFP expression (Fig. 5Go).



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  		Figure 5. Hybrid MSCV/HIV vector can transduce CD34+ cells in the absence of cytokine stimulation. Isolated UBC CD34+ cells were transduced immediately or were cultured for 2 days with growth factors prior to transduction. Transduced cells were washed, cultured with growth factors for 5-7 days, and analyzed by FACS for EGFP fluorescence. Results are representative of five or more independent experiments.

 
Stable Transgene Expression in Human Hematopoietic Progenitor Cells
To verify that very early human progenitor cells can be transduced by the hybrid LTR lentivirus, long-term bone marrow culture was performed. Transduced CD34+ cells were plated onto primary human stromal cells and maintained for 5-8 weeks. FACS analysis of these cells demonstrated that the percentage of EGFP+ cells remained constant (Fig. 6Go). Furthermore, the EGFP expression remained 1.5 to 2 logs over control fluorescence. These cells were also plated on methylcellulose to detect the progeny of long-term culture initiating cells (LTC-IC). After 10-12 days in culture, numerous EGFP+ colonies were detected using UV fluorescent microscopy. The percentages of colonies that are EGFP+ were similar to the FACS percentages (Table 3Go). These findings are consistent with transduction of very early hematopoietic precursor cells with no evidence of promoter shutdown of the hybrid LTR.



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  		Figure 6. Hybrid-based vector maintains the number of cells and the level of expression in transduced CD34+ cells. UCB CD34+ cells were isolated and stimulated with SCF, TPO, and flt-3 for 2 days. The cells were transduced with the hybrid virus, cultured on irradiated human stromal cells for 5 weeks, and then analyzed by FACS for EGFP fluorescence. Results are representative of three independent experiments.

 

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  		Table 3. Long-term transduction efficiency
 
Stable Transgene Expression in NOD/SCID Repopulation Assay
To confirm transduction of very early progenitor cells, the hybrid vector was tested in a repopulation assay. CD34+ UCB cells that were stimulated with SCF, TPO, flt-3 for 2 days, and then transduced by spinoculation. Analysis of the pre-injected CD34+ cells revealed that 20% were EGFP+ (data not shown). After infection, 2.0 x 106 UCB cells were immediately injected via tail vein into sublethally irradiated male NOD/SCID mice. At week 5 post-injection, mice were sacrificed and the BM cells were isolated and analyzed by FACS for the presence of human CD45+ and EGFP+ cells. Of the engrafted human CD45+ cells, 20% were EGFP+ (Fig. 7Go) similar to the pre-injection levels. Furthermore, the EGFP expression was 1.5 logs over control fluorescence. These findings are consistent with transduction and stable expression of the transgene in repopulating cells.



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  		Figure 7. Hybrid-based vector transduces NOD/SCID repopulating cells. UCB CD34+ cells were isolated and stimulated with SCF, TPO, and flt-3 for 2 days. The cells were transduced with the hybrid virus and immediately injected into sublethally irradiated NOD/SCID mice. At week 5, mice were sacrificed and the BM cells analyzed by FACS for human CD45+ and EGFP+ cells. % represents the percentage of human CD45+ cells that are also EGFP+.

 

   DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Viral Concentration Increases...
 Discussion
References
 
We have engineered a novel lentiviral vector with a hybrid MSCV/HIV LTR that successfully weds the high transgene expression of the MLV-based vectors with the high transduction efficiency of lentiviral vectors. To our best knowledge, this is the first report that demonstrates that a promoter can be functional when embedded in the HIV LTR as has been demonstrated for the oncoviral retrovirus vectors. In human CD34+ cells, the hybrid vector expresses its transgene 10- to 100-fold over the original HIV-based vector. This high level of expression and the percentage of EGFP+ cells were maintained over 8 weeks in LTC-IC assays and 5 weeks in NOD/SCID repopulation assay, indicating that the hybrid vector transduces very early hematopoietic precursors and that promoter shutdown does not occur. Thus, this vector is a valuable research tool for high and stable expression of transgenes in human hematopoietic progenitor cells.

Similar to the original HIV-based vector, the hybrid vector was able to transduce nondividing HeLa cells, an expected finding since all known HIV viral proteins that promote transduction of nondividing cells [7, 28-31] are present. The hybrid vector also transduced human CD34+ hematopoietic cells with greater efficiency than the MSCV-based vector when assayed under identical transduction conditions with normalized viral titers. The transduction efficiencies were 1%-4% and 10%-30% for the MLV-based and hybrid-based viruses, respectively. The efficiency for the hybrid-based virus decreased to 2%-10% in the absence of exogenous growth factors. While these numbers are less than those reported by others, the differences probably represent different experimental conditions [1, 2, 4]. For example, we did not use fibronectin or multiple exposures to viral supernatant. We transduced the CD34+ cells only once 18-24 hours after isolation. In contrast, Evans et al. transduced CD34+ cells twice within 24 hours of isolation [1]. These experimental differences and not the inherent differences in the viral vectors are the probable explanation for the differences in the reported transduction efficiencies. Further supporting this premise are the markedly different transduction efficiencies (10% versus 54%) that are seen in unstimulated CD34+ cells transduced with the same HIV-based vector [2, 4].

Although lentiviral vectors represent promising tools for gene therapy, numerous issues need to be addressed prior to clinical trial. Many of the current advances in vector design deal with the biosafety issue and have led to the second [31] and third generations [32] of the HIV-based vectors. The third generation vector has a deleted 3' LTR (self-inactivating-vector) which in theory will decrease the risk of expression of oncogenes downstream of the viral insertion site, although such an event has yet to be reported in human studies using retroviral vectors with intact LTRs.

More recent advances in lentiviral vector design deal with the poor expression of the transgene in human hematopoietic cells. This has important implications in using lentiviral vectors as therapeutic and research tools. Both the first [18] and second generation vectors contain an internal CMV promoter that does not express well in hematopoietic cells. The initial third generation vector [32] replaced the internal CMV promoter with the phosphoglycerate kinase gene (PGK) promoter but a recent study indicates that this vector does not express well in hematopoietic cells [33]. The addition of a post-transcriptional regulatory element of woodchuck hepatitis virus (Wpre) [34] increases the expression of the transgene to the same level as our hybrid vector [33, 35]. Interestingly, Wpre decreases the expression of the transgene off the human elongation factor EF1{alpha} promoter, suggesting that the effects of Wpre can depend on the promoter. Studies are ongoing to determine if the Wpre can further increase the expression of our hybrid vector.

The relatively weak activity of the PGK promoter in the absence of the Wpre is similar to the modest increase in expression seen in our vectors in which the internal CMV promoter was replaced with the RSV or MSCV promoter. A modest increase in expression has been reported in T cells using an HIV-based vector in which the CMV internal enhancer/promoter was replaced with an internal MLV LTR enhancer/promoter [21]. The increased expression probably indicates that the network of transcription factors and coactivators in hematopoietic cells favors the expression of the leukemia viral LTR over the immediate promoter of CMV, an expected finding considering that leukemia virus is specialized to infect hematopoietic cells while CMV infects a wider variety of cell types. Understanding which of the many potential DNA elements of the LTR contributes to the increased expression will lead to further improvement in the design of the enhancer/promoter for higher expression in hematopoietic cells.

The expression of the transgene is further increased when the internal MSCV enhancer/promoter is moved to the LTR. Three possible mechanisms for the increased expression of the hybrid vector include: A) loss of promoter interference from the HIV LTR [21]; B) duplication of the MSCV enhancer leading to synergistic increases in the activity of the 5' LTR, and C) utilization of the mRNA splicing sites that are 5' to the internal enhancer/promoter site—since splicing is linked to nuclear export, transcripts driven of the 5' LTR will be spliced and more efficiently exported out of the nucleus. While these mechanisms may contribute to the increase in expression, they do not completely explain the greater than 10-fold increase. Promoter interference appears to decrease expression by only two- to threefold [21]. Increasing the number of copies of the MSCV enhancers (2, 3, and 4 copies) at the internal site did not increase the expression of the transgene (data not shown). Splicing of the HIV viral transcript is considered inefficient, contributing to the nuclear entrapment of the transcript in the absence of the Rev protein [36]. Although the actual mechanisms for the increased expression remain unidentified, our results predict that some promoters will be more active when placed in the LTR versus at an internal site. This approach would be useful in designing lentiviral vectors for restricted expression in specific cell lineages, especially in cases in which Wpre decreases the expression of a particular promoter.


   ACKNOWLEDGMENTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Viral Concentration Increases...
 Discussion
 References
 
This work was supported by grants from the National Institutes of Health (CA75330) and the McCabe Fund, Philadelphia, PA.

A.M.G. is a Distinguished Clinical Scientist of the Doris Duke Charitable Foundation.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Viral Concentration Increases...
 Discussion
 References
 

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Received January 18, 2001; accepted for publication February 19, 2001.




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