INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

One method of transferring therapeutic genes into human cells for disease applications involves inserting the gene into a virus genome and letting the modified virus infect the cells. Because retroviruses integrate their genomes into the target cell chromosomes, retrovirus-mediated gene transfer theoretically provides for long-term expression of the therapeutic gene in the transduced cell. However, the retroviral preintegration complex does not traverse the nuclear membrane pore, requiring target cell division in order for the viral genome to be integrated (27, 44). Lentiviruses also integrate their genomes but do not require target cell division, since the lentivirus preintegration complex can traverse the nuclear membrane pore (8, 26). Therefore, lentiviruses are attractive vehicles for gene therapy applications requiring long-term expression of a therapeutic gene in nondividing cells (for reviews, see references 7 and 47).

Gene transfer systems based on human immunodeficiency virus type 1 (HIV-1) are by far the most developed lentivirus systems, with documented in vivo transduction of rat brain (5, 37, 38), retina (34, 36), muscle and liver (23), and mouse trachea (22). Mouse pancreatic islets were transduced ex vivo and transplanted in vivo, with stable expression of the transgene (17). In addition, HIV-1-derived vectors have transduced human corneal tissue ex vivo (50). Moreover, unstimulated human hematopoietic stem cells (HSCs) transduced in vitro have developed into mature T (14) and B cells (35) in in vivo models of lymphocyte maturation. Gene transfer systems have been derived from other lentiviruses as well, including HIV-2, simian immunodeficiency virus, feline immunodeficiency virus, (13a, 42), equine infectious anemia virus (33, 40), and visna virus (4a).

Although HIV-1 vectors are the most well studied, some authors have suggested that HIV-based vectors pose safety risks for human clinical applications (7, 47). The possibility has been raised that if the HIV-1 vector recombines with endogenous human retroviruses present in the cells (29, 32) or with exogenous viruses present during transient infections, there is a chance of generating replication-competent HIV-1 or transferring the vector to other cells in the patient. Such a recombination event is less likely to occur for nonhuman or even nonprimate vectors. In addition, even if the nonprimate lentivirus were to become replication competent, it may not be as destructive in humans as HIV-1, although the point has been debated (47).

Bovine immunodeficiency virus (BIV) is a lentivirus which infects cows and causes AIDS-like disease after a variable asymptomatic phase (19). Although the virus has a set of accessory genes similar to those of HIV and simian immunodeficiency virus, it is the most ancient known lentivirus and does not readily infect T cells. Instead, the virus is found predominantly in monocytes and splenic macrophages in vivo (19). In this report we describe the generation and characterization of gene transfer systems based on BIV clone 127 (18). The vectors were observed to transduce proliferating and nondividing human and nonhuman cell lines and primary cells, in some instances with virus titers almost as high as that of an HIV-1 gene transfer system (14).

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Plasmids. All restriction endonucleases were purchased from Roche Molecular Biochemicals (Indianapolis, Ind.). Plasmid pBIV, containing BIV proviral clone 127 (18), was obtained from the National Institutes of Health, Rockville, Md. Plasmid pCI was obtained from Promega (Madison, Wis.). Plasmid pCIGL contains the vesicular stomatitis virus glycoprotein (VSV-G) cDNA (9, 51) in the pCI polylinker, downstream of the human cytomegalovirus (CMV) immediate-early promoter and chimeric intron and upstream of the simian virus 40 (SV40) late polyadenylation signal. Plasmid pCrev, containing the HIV-1 rev cDNA under control of the CMV promoter, has been described previously (30). Plasmid pBH1 was generated by sequential insertion of two pBIV segments into the pCI polylinker using standard techniques: a 5.5-kb SmaI-XbaI fragment containing the gag, pol, vif, vpw, and vpy genes, as well as the first coding exons of the tat and rev genes; and a 1.3-kb DraIII-PvuII fragment containing the putative rev-response element (RRE) and the second coding exons of the tat and rev genes. Plasmids pBH2 and pBH3 were constructed in the same way, but the chimeric intron was deleted; moreover, the pBH2 5' BIV fragment also contained the 3' 70 bp of the leader, including the major splice donor site. Plasmids pBH1 and pBH3 were also modified by insertion of (1) an approximately 500-bp fragment of HIV-1 containing the RRE, (2) an internal ribosome entry site (IRES) from encephalomyocarditis virus (21), and (3) the puromycin N-acetyltransferase cDNA (49). Plasmid pBBB was generated by digesting plasmid pBIV with BfrI and BglII to remove most of the coding region and inserting into the gap a short polylinker created by annealing oligonucleotides BB5 (5'-TTAAGATTTAAATACGCGTGCGGCCGCA-3') and BB3 (5'-GATCTGCGGCCGCACGCGTATTTAAATC-3').

Immortalized cells. 293T cells were obtained from Gary Nolan (Stanford University, Palo Alto, Calif.). CEMSS cells were obtained from the AIDS Reagent Program (Rockville, Md.). A-10 and D-17 cells were obtained from the American Tissue Type Collection (Manassas, Va.). MN9D cells (13) were obtained from Rainer Ortman (Novartis, Basel, Switzerland). Embryonal rabbit epithelial (EREp) cells (39) were obtained from the National Institutes of Health. Human umbilical vein endothelial cells (HUVEC) (20) and primary rat aorta (smooth muscle) cells were obtained from Clonetics (San Diego, Calif.). HUVEC were cultured in EBM basal medium with the FGM bullet kit containing 0.1% human epidermal growth factor, 2% fetal calf serum (FCS), 0.4% bovine brain extract with heparin, 0.1% gentamicin-amphotericin B, and 0.1% hydrocortisone. Rat aorta cells were maintained in EBM basal medium with the EGM-MV bullet kit containing 0.1% human epidermal growth factor, 5% FCS, 0.4% bovine brain extract with heparin, 0.1% gentamicin-amphotericin B, 0.1% hydrocortisone and cultured in Primaria tissue culture flasks (Becton Dickinson Biosciences, San Jose, Calif.). CEMSS cells were cultured in RPMI 1640 medium supplemented with 10% FCS. All other cell lines were cultured in Dulbecco's modified Eagle medium supplemented with 10% FCS. HUVEC (1.25 × 10⁵) were suspended in 5 ml of medium and irradiated with 8,000 rads from a ¹³⁷Cs source irradiator (J. L. Shepherd, San Fernando, Calif.) and then transferred to a six-well dish and incubated for 2 days to allow for synchronization in the G₂/M phase of the cell cycle.

Virus production. 293T cells (4 × 10⁶ to 10 × 10⁶ 293T cells) were seeded into 10-cm-diameter dishes overnight and transfected the next day with 20 to 30 µg of plasmid DNA by the calcium phosphate method (Clontech, Palo Alto, Calif.). Typically, 20 µg of the vector, 10 µg of the packaging construct, and 3 µg of the VSV-G plasmid were used. In cases where the HIV-1 rev protein was required, 4 µg of plasmid pCrev was added. After 24 to 72 hr, the cells or the virus-containing medium was collected and analyzed in a variety of ways (see below). To assess the efficiency of transfection, a portion of the cells were analyzed for eGFP expression by flow cytometry, using a FACScan apparatus (Becton Dickinson Biosciences). To measure the amount of virus shed into the medium, the medium was cleared of cellular debris by low-speed centrifugation, and then 10 µl was lysed and analyzed for reverse transcriptase (RT) activity using a commercial kit (Roche Molecular Biochemicals).

Analysis of RNA levels: Northern blotting. Transfected cells were lysed and cytoplasmic RNA was prepared using a commercial kit (Qiagen, Valencia, Calif.). In addition, virus-containing medium was collected, subjected to low-speed centrifugation to remove cellular debris, and then subjected to high-speed centrifugation (50,000 × g for 90 min at 4°C) to collect the virus particles. The viral pellet was lysed and the viral RNA was prepared using a commercial kit (Qiagen). A fixed amount of cytoplasmic RNA (10 µg) or viral RNA (one-third of the RNA preparation, not quantitated) was subjected to 1% agarose gel electrophoresis and transferred to a nylon filter (Bio-Rad, Hercules, Calif.). The filter was exposed to 40 × 10⁶ cpm of a DNA fragment random primed with [³²P]dCTP using a commercial kit (Ambion, Austin, Tex.) and then washed and analyzed for bound probe with a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.). The probes included a BIV gag fragment and an HIV-1 gag fragment (both ~1 kb), an ~330-bp BIV U3 fragment, and an ~800-bp eGFP fragment.

Analysis of protein levels: Western blotting assay. Transfected cells were lysed on ice for 1 h in a buffer containing 1% NP-40, 150 mM NaCl, 10 mM Tris-Cl (pH 7.4), 1 mM EDTA, and Pefabloc protease inhibitor (Roche Molecular Biochemicals). The lysate was subjected to centrifugation at 8,000 × g for 20 min at 4°C to remove precipitated proteins and other debris. Alternatively, virus-containing medium was collected, subjected to brief centrifugation to remove cellular debris, and then subjected to high-speed centrifugation (50,000 × g for 90 min at 4°C) to collect the virus particles. The viral pellet was lysed directly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer (Novex, San Diego, Calif.). A fixed amount of cell or viral lysate was subjected to acrylamide gel electrophoresis and transferred to a nitrocellulose filter. The filter was exposed to rabbit serum specific for BIV Gag protein (obtained from the National Institutes of Health), then to horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin (Ig) antibody (Zymed, South San Francisco, Calif.), and then to the HRP substrate o-phenylenediamine dihydrochloride (OPD; Sigma, St. Louis, Mo.). Prestained molecular weight standards (Bio-Rad) were used to determine the approximate molecular weight of the BIV Gag bands. For the HIV Western blot, the filter was exposed to biotinylated anti-HIV Gag antibody (Beckman Coulter, Fullteron, Calif.) and then to steptavidin-conjugated HRP (Beckman Coulter) before the OPD reaction. For the HA Western blot, the filter was exposed to a biotinylated anti-HA antibody (Roche Molecular Biochemicals) and then to the steptavidin-conjugated HRP before the OPD reaction. For the VSV-G Western blot, the filter was exposed to mouse anti-VSV-G monoclonal antibody (MAb) (Sigma) and then to an HRP-conjugated goat anti-mouse Ig antibody (Zymed) before the OPD reaction.

Analysis of transduction. To transduce cells, the virus-containing medium was subjected to brief centrifugation to remove cellular debris and then 1 ml was added to fresh cells in polypropylene tubes (5 × 10⁵ CEMSS cells) or in six-well dishes (3 × 10⁵ to 6 × 10⁵ adherent cells seeded per well the previous day). Protamine sulfate (Sigma) was added to the wells at a final concentration of 8 µg/ml and the tubes or dishes were subjected to centrifugation ("spinoculation") at 1,500 × g for 2 to 3 h at 32 to 37°C. In later experiments, the tubes or wells were also supplemented with 10 mM HEPES buffer prior to spinoculation to prevent the pH of the medium from rising while the cells were in the centrifuge. After spinoculation, the tubes and dishes were processed in different ways: supernatant was aspirated from the tubes, and the CEMSS cells were suspended in fresh medium and transferred to six-well dishes. In contrast, the spinoculated dishes were placed back into the incubator for 30 to 60 min, and then the medium was removed and fresh medium was added to the wells. At 2 to 3 days postspinoculation, a portion of the cells were removed from the plate and analyzed for eGFP expression by flow cytometry, using a FACScan apparatus (Becton Dickinson Biosciences). For virus preparations containing the vectors pBC3MP and pBC3MPsar, 293T cells were subjected to spinoculation with serial dilutions of the virus-containing medium, and fresh medium supplemented with puromycin (5 µg/ml) was added to the cells 24 h postspinoculation. Seven to ten days later, colonies were counted by direct visualization.

Infection of primary T cells. Human peripheral blood mononuclear cells (PBMC) were isolated from adult peripheral whole blood by Ficoll density gradient centrifugation, rinsed in phosphate-buffered saline, and suspended in RPMI 1640 medium supplemented with 10% FCS. A portion of the PBMC were activated by adding interleukin 2 (Peprotech, Rocky Hill, N.J.) to the medium at a final concentration of 200 U/ml and culturing the cells for 3 days in 12-well dishes (3 × 10⁶ cells per well) precoated as follows: the dishes were incubated with 1 µg of goat anti-mouse Ig Fc (Pierce, Rockford, Ill.) per ml for 3 h, rinsed with phosphate-buffered saline, incubated with a mixture of MAbs (1 µg of anti-CD3 [OKT3] per ml and 10 ng of anti-CD28 [BD PharMingen, San Diego, Calif.] per ml) for 1 h and rinsed with medium. Activated or unstimulated PBMC (5 × 10⁵) were spinoculated with viral supernatant in polypropylene tubes similar to CEMSS cells (see above); after spinoculation, the cells were rinsed and suspended in medium containing interlenkin-2 and cultured in 24-well dishes precoated with the anti-CD3 and anti-CD28 MAbs. Four days and 2 weeks later, a portion of the cells was removed from the well and analyzed for eGFP expression by flow cytometry, using a FACScan apparatus (Becton Dickinson Biosciences). T cells were identified by light scatter properties and by expression of CD4 and CD8, using allophycocyanin (APC)-conjugated anti-CD4 and PerCP-conjugated anti-CD8 MAbs (Becton Dickinson Biosciences).

Infection of HSCs. Human CD34⁺ cells were isolated from granulocyte colony-stimulating factor-mobilized peripheral whole blood from healthy donors using Isolex 300SA (Baxter Healthcare, Deerfield, Ill.). The cells (80 to 90% pure CD34⁺) were aliquoted (10⁷) and frozen in medium consisting of 45% Iscove's modified Eagle medium, 45% FCS and 10% dimethyl sulfoxide. Prior to transduction, the frozen cells were first thawed in buffer containing 2% FCS, 1% HEPES, and 10 U of heparin per ml. After thawing, the cells (5 × 10⁵) were either spinoculated (see above) with viral supernatant in the absence of cytokines or cultured for 48 h in cytokine-containing medium (X-vivo15 medium [BioWhittaker, Walkersville Md.], thrombopoietin [tpo] mimetic [50 ng/ml; Novartis], flt3 ligand [100 ng/ml], and c-kit ligand [100 ng/ml; both from Systemix, Palo alto, Calif.]), and then spinoculated with viral supernatant. After infection, the cells were cultured in cytokine-containing medium. Three days or 2 weeks later, a portion of the cells was stained with APC-conjugated anti-CD34 antibody (Becton Dickinson Biosciences) and analyzed for eGFP on the CD34⁺ cells on a FACScan apparatus (Becton Dickinson Biosciences).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Can BIV transduce human cells? To determine whether BIV could transduce human cells, the wild-type BIV genome (Fig. 1A) was modified by insertion of the eGFP marker gene, producing construct BCG. The eGFP gene was inserted into an interior position within the viral envelope gene so as not to affect viral rev or tat expression or RRE function. The human kidney carcinoma cell line 293T was cotransfected with construct BCG and a plasmid encoding VSV-G; 2 days later, the virus-containing medium was collected and exposed to fresh 293T cells. In addition, the medium was added to the embryonal rabbit epithelial cell line EREp, which supports wild-type BIV replication (39). Three days later, the exposed cells were assayed for eGFP expression by flow cytometry (Fig. 1B). A subset of the 293T cells (approximately 5%) expressed eGFP, indicating that BIV could carry out all of the functions (including reverse transcription and integration) required for transduction of human cells. Similar transduction efficiencies were noted for the EREp cell line (Fig. 1B).

View larger version (43K): [in this window] [in a new window]   FIG. 1.   (A) Wild-type BIV genome. Viral coding regions are arranged in three horizontal lines representing the different reading frames. The rev and tat genes are composed of two coding regions each. The first coding region of rev and the tmx coding region are in the same reading frame as env. The approximate position of the insertion of CMV-eGFP (in construct BCG) is indicated above the top line. (B) BIV can transduce human cells. VSV-G-pseudotyped construct BCG was produced in 293T cells and exposed to EREp (right column) and fresh 293T (left column) cells; 3 days later, the cells were analyzed for eGFP expression by flow cytometry. The percentage of eGFP+ cells is indicated inside each histogram.

BIV packaging constructs. As a first step towards setting up a BIV-based gene transfer system, in which the eGFP gene and viral genes are encoded on separate plasmids, packaging constructs (containing the viral genes) were generated (Fig. 2A) and assayed for gag mRNA expression (Fig. 2B) and virus production (Table 1) in the transient expression system. One construct, BIVC, is identical to wild-type BIV except for a precise replacement of the 5' LTR U3 region with the human CMV immediate-early promoter. The junction between the two segments is located at the identical TATA boxes to maximize the chances that the viral RNA would contain the proper 5' end for infection of target cells. The three other BIV packaging constructs contain the CMV promoter and transcription initiation site linked to the BIV leader near of the gag gene, deleting the 5' LTR and primer binding site andin the case of constructs BH1 and BH3the major splice donor. Construct BH1 contains a small chimeric intron inserted between the CMV and BIV segments. Downstream of the pol gene, construct BH2 contains all viral coding sequences except for a deletion (approximately 1 kb) of the interior of env (not predicted to affect tat and rev expression or RRE function), while constructs BH1 and BH3 contain BIV sequences terminating approximately 250 bp downstream of the pol gene. Since BH1 and BH3 lack the rev gene and the RRE, the HIV-1 RRE was inserted downstream of the BIV sequences and HIV-1 rev protein was provided in trans when the constructs were characterized. In addition, these two constructs contain the puromycin N-acetyltransferase cDNA (49) coupled to an IRES from the encephalomyocarditis virus (21), for selecting cell lines stably producing the packaging construct.

View larger version (28K): [in this window] [in a new window]   FIG. 2.   BIV packaging constructs and expression in transfected 293T cells. (A) wild-type BIV, CMV-driven BIV, and three packaging constructs (BH1 to -3) are depicted for their gag, pol, and env genes; accessory genes are not shown. Also depicted are the viral major splice donor (MSD), a small chimeric intron (in), the HIV-1 RRE, the puromycin N-acetyltransferase cDNA linked to an IRES from encephalomyocarditis virus (IRES-puro), and the SV40 late polyadenylation signal (SV40 polyA). (B) Northern analysis of the constructs' steady-state cytoplasmic expression levels. Cytoplasmic RNA from transfected 293T cells was probed for gag sequences; the high-molecular weight band in each lane corresponds to the construct's gag mRNA.

View larger version (31K): [in this window] [in a new window]   FIG. 3.   BIV produces levels of virus similar to HIV-1. BIV packaging construct BH2 and an HIV-1 packaging construct were each modified by insertion of HA tags at the C terminus of Gag and deletion of pol (see text). Parental and modified viruses were produced in 293T cells, collected and subjected to anti-HIV-1 Gag (lanes 1 and 2), anti-BIV Gag (lanes 3 and 4), or anti-HA (lanes 5 and 6) Western blotting. Lane 1, parental HIV-1 packaging construct. Most of the Gag precursor polyprotein (PrGag) has been cleaved. Lane 2, modified HIV-1 packaging construct. The amount of uncleaved Gag polyprotein is similar to the amount of capsid protein (CA) in the parental virus preparation, indicating that the modifications did not alter virus production substantially. Lane 3, parental BIV packaging construct. Lane 4, modified BIV packaging construct. As with the case of the HIV-1 packaging construct, the modification did not alter virus production. Lane 5, modified HIV-1 packaging construct. Lane 6, modified BIV packaging construct, after normalization by RT assay of the parental constructs. The amount of Gag precursor polyprotein is similar to the amount in lane 5, indicating that the RT assay has comparable sensitivity for BIV and HIV-1 virus and hence, that the packaging constructs produce similar levels of virus.

BIV vectors. Next, BIV-derived vectors were generated, containing the eGFP marker gene and all viral elements required in cis for transfer into target cells. A panel of vectors was generated (Fig. 4), differing in (i) the amount of gag and env sequences (BC versus BC2 prefix), (ii) the internal promoter driving eGFP expression: CMV (constructs with a CG suffix) versus PGK (1) (constructs with a PG suffix) versus MND (modified myeloid proliferative sarcoma virus promoter [43]) (constructs with an MG suffix); (iii) the placement of eGFP within the vector (i.e., upstream or downstream of the putative BIV RRE) (BC2 versus BC3 prefix); and (iv) additional segments inserted downstream of the putative BIV packaging signal (constructs with gag, ppt, and sar suffixes). In addition, two vectors contained a modified 3' LTR in which a large portion of U3 (including the TATA box) was replaced by a small SV40 segment containing the late polyadenlyation signal USE (45) (BC3 versus BC4 prefix).

View larger version (56K): [in this window] [in a new window]   FIG. 4.   BIV vectors and transduction efficiencies. All vectors contain the CMV immediate-early promoter, ending in the TATA box, linked to the BIV 5' LTR starting immediately after the TATA box. BIV sequences terminate at the gag start codon (BCCG and BCPG) or approximately 510bp into the gag coding region (all others). All vectors contain the eGFP cDNA linked to a heterologous, "internal" promoter: CMV, PGK, or MND (see text). Some vectors contain one or more insertions between the BIV 5' segment and the internal promoter: these insertions include a potential BIV cPPT, the 3' segment of the gag gene, the beta interferon SAR, and the putative BIV RRE. Downstream of the eGFP cDNA lies the BIV 3' LTR and approximately 130 bp (BCCG and BCPG), 1.2 kb (BC2CG, BC2PG, and BC2MG) or 80 bp (all others) of adjacent env sequences. Two vectors (BC4MG and BC4MGppt) contain modified 3' LTRs in which most of the 3' LTR U3 region has been replaced by the SV40 late polyadenylation signal enhancer element (SINSV). Transcription start sites and directions are indicated with arrows. At the bottom are depicted two HIV-1 control vectors used in this study. At the right are the transduction efficiencies of the vectors in 293T cells 3 days postinfection, using packaging construct BH2 and VSV-G, in a series of experiments. Infections from experiments 3 to 5 were performed in the presence of an additional buffer to retard pH elevation during spinoculation. As a result, transduction efficiencies were elevated.

View larger version (33K): [in this window] [in a new window]   FIG. 5.   Northern analysis of BIV RNA in transfected 293T cells (left) and in virus particles shed from the transfected cells (right). Cytoplasmic RNA was probed with BIV sequences at the 3' end of the genome, to detect all packaging construct and vector RNAs. Viral RNA was probed with eGFP sequences, to detect full-length vector RNA and RNA initiating at the internal promoter. Lane 1, vector BC2PG; lanes 2 and 7, packaging construct BH2; lanes 3 and 8, BH2 and BC2PG; lanes 4 and 9, BH2 and BC2CG; lanes 5 and 10, BH2 and BCPG; lanes 6 and 11, BH2 and BCCG. The positions of certain RNAs are indicated: gag, the gag mRNA encoded by BH2; BC2F, full-length BC2PG and BC2CG vector mRNA; BC2I, internally initiated BC2PG and BC2CG vector mRNA; BCF, full-length BCPG and BCCG vector RNA; BCI, internally initiated BCPG and BCCG vector mRNA; BC2S, spliced BC2PG and BC2CG vector mRNA. Note that this last RNA is only present in the BC2 vectors, since the rev splice acceptor is present in the BC2 vectors but not the BC vectors.

View larger version (24K): [in this window] [in a new window]   FIG. 6.   Analysis of virus concentration and the duration of transgene expression in transduced human cell lines. A BIV preparation consisting of packaging construct BH2, VSV-G, and vector BC3MG was collected by centrifugation, then suspended in the original volume (1× virus) or in a 10-fold-lower volume (10× virus) and exposed to fresh 293T or CEMSS cells. eGFP expression was measured at 3 and 16 days postinfection by flow cytometry; the percentage of transduced cells is indicated in each histogram.

Transduction of other cell lines and nondividing cells. Concentrated BIV, prepared in 293T cells using packaging construct BH2, vector BC3MG, and VSV-G, was used to transduce a panel of cell lines: D-17, a dog osteosarcoma line; A-10, a rat smooth muscle cell line; HUVEC, a human endothelial cell line; and MN9D, a mouse neuronal cell line. In each of these cell lines, the BIV preparation transduced a large percentage (63 to 88%) of the cells, even 2 weeks postinfection (Fig. 7). In addition, primary rat endothelial cells were transduced by BIV, albeit not as efficiently as the immortalized lines (22%).

View larger version (35K): [in this window] [in a new window]   FIG. 7.   BIV can transduce a variety of cell lines and primary cells. A 1× (D-17 cells) or 10× (all others) BIV preparation consisting of packaging construct BH2, VSV-G, and vector BC3MG was exposed to the indicated cell lines or primary cells (see text for details on each cell type). Two weeks later, the cells were analyzed for eGFP expression by flow cytometry; the percentage of transduced cells is indicated in each histogram. For comparison, the HIVPG histograms indicate the cells exposed to an unconcentrated HIV-1 preparation consisting of a packaging construct, VSV-G, and a vector containing the PGK internal promoter.

View larger version (41K): [in this window] [in a new window]   FIG. 8.   BIV can transduce nondividing cells. A 10× BIV virus preparation consisting of packaging construct BH2, VSV-G, and vector BC3MG was exposed to HUVEC 2 days after the cells were irradiated to abrograte cell division (right column) or to nonirradiated HUVEC (left column). Two days later, the cells were analyzed for eGFP expression by flow cytometry; the percentage of transduced cells is indicated in the histogram (right column). For comparison, the HIVCG histograms indicate cells exposed to an unconcentrated HIV-1 preparation consisting of a packaging construct, VSV-G, and a vector containing the CMV internal promoter. In addition, the MLV histograms indicate cells exposed to an MLV preparation consisting of a packaging construct, VSV-G, and an LTR-driven eGFP vector.

View larger version (39K): [in this window] [in a new window]   FIG. 9.   BIV can transduce resting human lymphocytes. Unstimulated human PBLs were infected with 10× and 40× BIV consisting of packaging construct BH2, VSV-G, and vector BC3MG. After infection, the cells were activated and cultured for 4 days (left column) or 2 weeks (right column) in the presence of antibodies specific for CD3 and CD28, after which a portion of the cells were analyzed for eGFP expression by flow cytometry. The percentage of transduced cells is indicated in each histogram. For comparison, the cells were infected with the 10× HIV-1 PGK preparation.

View larger version (51K): [in this window] [in a new window]   FIG. 10.   BIV can transduce resting HSCs Unstimulated human HSCs were infected with 10x and 40x BIV consisting of packaging construct BH2, VSV-G, and vector BC3MG. After infection, the cells were activated and cultured for 3 days (left column) or 2 weeks (right column) in the presence of tpo mimetic, flt3 ligand, and c-kit ligand, after which a portion of the cells were analyzed for eGFP expression by flow cytometry. The percentage of transduced cells is indicated in each histogram. For comparison, the cells were infected with the 10× HIV-1 PGK preparation.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we have demonstrated that gene transfer systems derived from BIV can transduce a variety of cell types from a variety of organisms in vitro. Transduction was observed in cell lines and primary cells, including resting human HSCs, as well as irradiated, nondividing human endothelial cells. In several cell types, transduction efficiencies approached those of an HIV-1 gene transfer system (14), and in most cell types, transgene expression was stable for at least 2 weeks. In addition, the titer of VSV-G-pseudotyped BIV preparations could be increased by centrifugation of the virus. One of the unconcentrated VSV-G-pseudotyped BIV preparations possessed a titer of approximately 5 × 10⁵ infectious units (IU) per ml in the human kidney epithelial cell line 293T. With virus concentration, the titer could be raised much higher.

With these results, BIV is the third nonprimate lentivirus to be reported to efficiently transduce nondividing cells in vitro. VSV-G-pseudotyped gene transfer preparations derived from feline immunodeficiency virus were observed to transduce G₁/S-arrested human and nonhuman cell lines, as well as postmitotic human monocyte-derived macrophages and neurons; viral concentration yielded titers (in growing HeLa cells) of 1.8 × 10⁷ IU/ml (42). VSV-G-pseudotyped gene transfer preparations derived from equine infectious anemia virus were observed to transduce G₁/S-arrested human (40) and canine cell lines, as well as postmitotic primary rat neurons, with titers in the canine cells as high as 5 × 10⁶ IU/ml (33). In contrast, VSV-G-pseudotyped gene transfer preparations derived from visna virus transduce human and sheep cells poorly, due to blocks in reverse transcription and integration (4a).

Although the VSV-G-pseudotyped BIV gene transfer preparations transduced all of the human cells tested, the relative transduction efficiencies varied greatly. The epithelial cell line 293T, the T lymphoid cell line CEMSS and the endothelial cell line HUVEC were all transduced efficiently, although the percentage of eGFP⁺ cells declined over time in the 293T cells. In addition, although CEMSS cells were efficiently transduced, the same was not true for primary lymphocytes: BIV preparations transduced 16-fold fewer proliferating PBLs and 29 to 44-fold fewer unstimulated PBLs than did an HIV-1 preparation. This relative inefficiency is not surprising, in light of the BIV's lack of tropism for lymphocytes in vitro and in vivo (19). In contrast, BIV transduced HSCs moderately well, as a BIV preparation transduced only 2.5-fold fewer unstimulated HSCs than did the HIV-1 preparation.

In unstimulated human PBLs and unstimulated human HSCs, BIV transduced a portion of the cells in such a way as to express very low levels of eGFP, and only for a short duration (i.e., less than 2 weeks). One possible explanation for this phenomenon is pseudotransduction: eGFP protein expressed in the transfected cells is associated with the virus particles and transferred to the target cells during infection and then is diluted out of the cells as they divide. Pseudotransduction can occur with other transgenes besides eGFP and has been observed for VSV-G-pseudotyped MLV vectors (16, 28) and spleen necrosis virus vectors (J. Douglas and S. Tamaki, unpublished data). Other possible explanations for the transient eGFP phenomenon include eGFP expression from unintegrated forms of the vector which are degraded over time and transgene promoter inactivation occurring on integrated forms of the vector.

The HSCs infected with the 40× BIV contained, in addition to the population expressing very low levels of eGFP, a population which expressed high levels of eGFP. Despite being exposed to more virions, this population expressed slightly lower levels of eGFP per cell and was less stable over 2 weeks than the cells transduced with the 10× BIV. Since VSV-G protein is toxic to cells, it is possible that the 40× BIV contained levels of VSV-G that damaged the HSCs during infection.

The lower transduction efficiencies exhibited by BIV, relative to HIV-1, could be due to deficiencies during infection. For example, BIV RT or integrase might interact with human proteins less efficiently than do the HIV-1 counterparts. However, it is also possible that the BIV preparations contained fewer infectious virus particles than the HIV-1 preparations. Although the two viruses' packaging constructs were often found to produce comparable numbers of virus particles and to incorporate VSV-G with comparable efficiency, the extent of virion encapsidation of the full-length vector RNA and primer tRNA was not analyzed thoroughly. Preliminary experiments indicate that HIV-1 preparations contain up to 3.5-fold more full-length vector RNA than do BIV preparations (R. D. Berkowitz, unpublished data). In any regard, the transduction efficiencies exhibited by BIV should certainly rise as the titer of infectious virus is increased, i.e. by increasing virus production or the efficiency of vector RNA encapsidation. Such increases in titer should be particularly valuable for cell types that require a high multiplicity of infection for efficient transduction or high-level transgene expression.

In addition to increasing viral titer, future studies on BIV gene transfer systems should evaluate the possibilities of potentially harmful side effects of such systems, i.e., generation of replication-competent retrovirus and propagation by or recombination with HIV virions during natural HIV infection. BIV does not infect human cells, presumably due to a number of blocks including the lack of a competent viral envelope, but nevertheless BIV-transduced cells must be monitored closely with a sensitive replication-competent retrovirus detection assay. In addition, if an individual containing BIV-transduced cells is infected with HIV, it is theoretically possible that the HIV virions could spread the BIV vector to new cells and/or recombine with the vector. Although it is not known if such an event would be harmful, the likelihood of the event could be measured by in vitro cross-packaging (13a) and recombination studies.