Genetic diseases can occur in various ways with complex causes, but conventional therapies are very limited or non-existent in many instances. Gene therapy approaches attempting to subvert, correct or prevent the pathogenic mechanisms of specific genetic disease by the introduction of corrective genetic information into appropriate target living cells represent a good alternative. It has been over 30 years since the first proposal of using gene therapy as a therapeutic method for the treatment of genetically determined inherited diseases (21). The idea of treating human diseases by introducing functional genes into living cells to achieve therapeutic purpose has now become a clinical reality (3). The first successful attempt of using molecular-based therapeutic techniques for the treatment of human recessive hereditary deficiency was documented in 2000 when Cavazzana-Calvo and his co-workers demonstrated they were able to overcome severe combined immunodeficiency (SCID-X1), an X-linked inherited disorder in children, through retrovirusmediated transfer of complementary DNA containing a gamma c and ex vivo infection of CD34+ cells (8). Successful completion of this clinical gene therapy showed great promise for the use of gene therapy to provide full correction of genetic disease phenotype and associated clinical benefits. Today, human gene therapy represents a promising new form of medicine which is under active development. The potential application of gene transfer technology has now been extended to the treatment of a variety of diseases including infectious diseases, AIDS, neurological disorders and cancer (7, 20, 27, 35).
Successful gene therapy approaches are largely dependent on the development of safe and efficient gene-delivery systems to transport therapeutic genes into the target destination. The vectors, or gene delivery systems, play a crucial role as effective tools for genetic modification of the majority of somatic cells in vitro and in vivo in the development of human gene therapy protocols (12, 25, 42). Vectors derived from a variety of viruses including retroviruses, adenoviruses, adeno-associated viruses, baculoviruses and herpesviruses, are currently being developed and evaluated for their potential use as gene transfer vehicles (27). Among these viral vectors, genetransfer vectors based on lentiviruses, particularly HIV-1-derived vectors, have been widely used for gene therapy applications since their initial construction in 1991 (36) and the interest in using such systems in applied settings will continue to grow (18, 31, 46). Present studies have shown that HIV-1-based vectors are attractive gene delivery tools due to their relatively large coding capacity, efficient gene transfer, ability to establish long-lasting transgene expression, ability to integrate into genomes of nondividing cells and to inhibit wild type HIV-1 replication in the absence of any anti-HIV-1 insert (2, 4, 5, 11, 42). Because of the potential for possible clinical gene transfer applications in the future, lentiviral vectors mediated gene transfer have been tested for their ability to infect various types of cells in vitro, in vivo and ex vivo, including hepatocytes (19, 41), hematopoietic cells (1, 14), stem cells (30, 40), monocyte-derived dendritic cells (22), lymphocytes (13, 47), monocytes/macrophages (29, 34, 38), and neurons (33, 44). All these cells are important targets in human gene therapy (4, 25, 35).
To further explore the use of a lentiviral vector as a potential gene transfer tool in human gene therapy, it would be necessary and important to develop an in vitro transfection protocol for consistent production of high yields of vector virus. The objectives of this study are directed towards optimization of in vitro protocols for DLV vector production and vector mediated gene transduction. We have established a method for preparation of high-titer vector and vector concentration through a simple one-step ultracentrifugation. Constructed HIV-1-based vectors pseudotyped with a VSV-G envelope protein are highly infectious to human T-cell lines and cells derived from a variety of mammalian species. DLV-mediated gene delivery into human T-cells facilitate a stable and long-term transgene expression and transductant cells become refractory to the infection by wild type HIV-1, suggesting that the transduced cells are protected from HIV-1 infection. We also demonstrated that these vectors are likely to be safe to use since we detected no generation of replication-competent virus (RCV) through vector recombination in transduced cells.
The DLV pseudotyped with the VSV-G envelope protein used in this study was generated in 293T cells according to the well-established calcium phosphate co-precipitation method. Transfection was evidenced by the presence of large percentage of GFP+cells (>70%) at day 1 post-transfection which extended to the whole cell population by day 3. This was accompanied by rapid increase of vector yield from 5.6×106 IU/mL at day 1 to 10.7×106 IU/mL at day 3 (Fig. 1A). To define the vector production by time, vector generated from each post-transfection day was separately quantified. Although many vectors were assembled shortly after transfection (5-7.5 × 106 IU/mL at day 1), the majority of vectors were produced at day 2 (>10×106 IU/mL) as shown in Fig. 1B. Vector production dropped to less than 6×106 IU/mL at day 3. Because of the cellular fusion and detachment of large percent of transfected cells, vector production was dramatically reduced at transfection day 4 and subsequent days (data not shown).
Figure 1. DLV (DHIV-Rev-) production in 293T packaging cell line DLV vector was produced under the same transfection protocol and titration of vector by limited dilution infectivity assay using CEM cells. A: Accumulative DLV production measured at selected post transfection time. B: Comparative analysis of vector produced in cell-free medium supernatant and associated within the 293T packaging cells at different post transfection times. The result of each test represented an average of three experiments.
To define the localization of DLV, we comparatively analyzed daily production of DLV released into cell culture medium (cell-free vector) and vectors associated with the packaging cells (cell-associated vector). The majority of assembled DLV (>97.5%) was released in the cell-free form in the cell culture medium while only a small portion of vector ( < 2.5%) was associated with the packaging cells (Fig. 1B). This led to the exclusion of the cellular pellet to be used for vector isolation and concentration.
To generate high-titer vector stocks, we have established a one-step ultracentrifugation protocol to concentrate vector produced in large-scale or from different batches of viral transfection. As shown in Table 1, the vector virus was effectively concentrated using this method and concentrated vector yielded a final titer up to 9.83±2.25×108 IU/mL. This vector concentration method is relative simple, rapid, easily reproducible, and it allowed a high recovery rate of infectious vector (90%) (Table 1).
Table 1. DLV production in 293T cells and concentration by ultracentrifugation
All three DLV vectors were capable of infecting CEM cells and they shared the same pattern of transduction efficiency, which is largely related to the concentration of DLV stocks.
As shown in Fig. 2, approximately 30% cells were successfully transduced when a low-titer of DLV stock was used (MOI = 1.0). This transduction efficiency increased to about 60% at a MOI of 5 and reached over 80% at a MOI of 20, indicating that the transduction efficiency is directly correlated with DLV titer. More than 90% of CEM cells became GFP+ when a more concentrated DLV preparation (MOI = 50) was used for the transduction. In addition, human glioblastoma cells (HTB-14) available in this laboratory were also tested for their susceptibility for DLV transduction. We demonstrated that DLV were highly infectious to HTB-14 cells with a transduction efficiency of 50% at MOI of 1.0 and over 90% at MOI 20 (data not shown). This finding may suggest DLV have the ability to transduce human microglial cells, which are very important target cells of HIV-1 infection in the human central nervous system.
Infectivity of these DLV to other cell lines were determined and compared by the titration of the same DLV preparation in ten different cells derived from human and other mammalian species. All these cells appeared to be susceptible to DLV infection. However, efficiency of DLV-mediated gene transduction of these cells differed remarkably (>10 000 times). As shown in Fig. 3, human lymphoid cells (CEM, MT-2, SupT1 and HTB-14) were the most sensitive to DLV infection while HeLa, 293T and WI38 cells were comparatively less sensitive, and murine-derived NIH3T3 and PA317 cells were the least sensitive. The ability to infect a wide spectrum of cell types suggests the potential use of these DLV pseudotyped with VSV-G in gene transfer for human and other mammalian species.
Figure 3. Transduction of different cell lines with DLV. Cells were harvested at their exponential growth phase and adjusted to a concentration of 2.0×105 cells/mL. 1 mL of this cell preparation was pelleted down an Eppendorf tube and for each vector 6 tubes of this cell pellet were prepared. Master preparation of DLV was 10-fold serially diluted (10-1 to 10-6) with serum-free medium containing 8ug/mL Polybrene. Infection of cells with DLV was conducted by resuspending the prepared cell pellet with 1.0 mL DLV preparation from 10-1 to 10-6, separately. Following 1 h adsorption, infected cells was inoculated into a 96-well plate at a concentration of 0.1 mL/well for cells (2.0×104 cells/well) and 4 wells/dilution of vector for each cell line. The 96-well plate was covered with sensitive film and incubated at 37℃ with 5% CO2. At day 3 post infection, GFP+ cells were visually counted and documented under either normal or fluorescent light (Olympus IX 70) by using mounted Olympus digital camera and software MagnaFire. The infectivity of DLV to 11 cells to DLV was determined by comparing vector titer (average of GFP positive cells from 4 wells at the endpoint dilution).
Cloning culture of DLV-transduced CEM cells were conducted in 96-well plates using the limited dilution method. The GFP positive cell clones derived from individual transduced cells were cultured with conditioned medium prepared from normal CEM cultures. By days 10-14, clonal cultures formed sizable cell colonies, then were transferred to a 12-well plate for 7-10 days before propagated in TC-25 cm2 flasks. Clonal cells were subcultured more than 60 times in vitro in a period of 1.5 years and showed no change in GFP expression in transduced cells (Fig. 4A, fluorescence light; 4B, normal light). This long-term stable transgene expression was also demonstrated by analyzing nucleic acid extracted from transduced CEM cells by RT-PCR (Fig. 4C).
Figure 4. Stable long-term expression of GFP gene in DLV-transduced CEM cells. A: Photomicrograph of GFP+ transductant CEM cells at passage 60 under fluorescence light; B: Photomicrograph of GFP+ transductant CEM cells at passage 60 under normal light; C: RT-PCR detection of transgene (GFP) expression in transductant CEM cells. Lane 1: 1kb plus DNA ladder (Invitrogen); 2: Total RNA from the 9th passage DLV-transduced CEM; 3: Total RNA fron the 48th DLV-transduced CEM; 4: Untransduced CEM; 5: Negative Control (water); 6: Positive control (plasmid DHIV-Rev-). Gel running: 2% agarose, 50V 90min, EB staining, Bio-Rad FX molecular imager scanning. Target fragment size: 373bp.
The potential for the generation of RCV through vector recombination during vector production, transduction and long-term culture of transduced cells was investigated using both p24 ELISA and viral infectivity assays. We have detected no RCV in all the samples we tested (data not shown). Initially, we examined the conditioned media collected from both early (8-10 passages) and later (>40 passages) cultures of different clones of DLV-transduced CEM cells and p24 antigen was not detected in these clones over a period of 1 year. The medium samples were then tested for infectious virus by inoculating the media to several human T-lymphocytes including CEM, SupT1 and MT-2 cells. Following in vitro cultivation for at least one month, these cultures showed neither any GFP+cells nor p24 production. Over a dozen of the transduced cultures were analyzed and generation of RCV was not detected (Data not shown). In addition, the negative detection of RCV was not due to the use of lower DLV preparations since present study included the use of 5 different DLV stocks with an average vector titer of more than 109 IU/mL.
The lentiviral vector-mediated inhibitory effect on viral replication was demonstrated by challenging transduced CEM with a wild type HIV-1 strain (Fig. 5). When normal CEM was infected with HIV-1, viral replication was detected shortly ( < day 5) and followed by rapid increase of viral p24 production which was evidenced by the massive formation of typical ballooning-shaped cells (syncytia). Viral replication reached a peak by post infection day 15 and then slowed down gradually due to the death of affected cells. When the same HIV-1 stock was inoculated into a transduced (50% GFP+) CEM population, HIV-1 replication was partially inhibited. This limited antiHIV-1 infection was determined by the delayed detection of viral replication, reduced viral production (p24) and limited level of syncytial formation. In comparison, HIV-1 replication was completely inhibited when GFP+ clones were challenged with the same viral stock. Similar results were also obtained when these clones were challenged with a more concentrated HIV-1 dose (MOI = 1.0). Further analysis of these infected cells revealed that GFP+ clones showed a consistent high percentage of cell viability (>92%) during the course of infection. In contrast, HIV-1 infection resulted in significant drop of cell viability to less than 15% for control CEM at postinfection day 21 as compared to 60% for the mixed population (Fig. 6A). The percentage of GFP+ cells gradually increased from the initial 50% to 80% following HIV-1 infection while no change was observed for the uninfected population (Fig. 6B). These data clearly suggest the resistance of GFP+ cells to HIV-1 infection and possibly vector mobilization.
Figure 5. Inhibitory effect of DLV on HIV-1IIIB replication. Viral infection of normal CEM (CEM-N) was monitored and compared with the same infection conducted in both a DLV-transduced CEM clone (GFP+ clone) and a cell population composing of equal number of normal CEM and GFP+ cells. Data show HIV-1 replication was significantly suppressed in the mixed culture and completely inhibited in the GFP+ clone.
Figure 6. Comparative analysis of cell viability and percentage of a transduced CEM cell population (50% GFP+) infected (I) or Uninfected (UI) with HIV-1IIIB. A: Kinetics of cell viability of a 50% GFP+ CEM cells compared to normal CEM cells. B: Percentage of GFP+ cells gradually increased with HIV-1 infection compared to no change for the uninfected cell population. CEM/GFP-I = HIV-1 infected transducatant CEM (50% GFP+) cells, CEM-GFP-UI = Transductant CEM (50% GFP+) cells without HIV-1 infection, CEM-I = normal CEM cells infected with HIV-1.
To define vector mobilization to untransduced cells, partially transduced CEM cells containing 10%, 30% and 50% GFP+ cells were infected with a low dose of HIV-1 (MOI = 0.01). We observed that the initial percentage of GFP+ cells increased very slowly for the 10% GFP+ population but rapidly increased for the 50% GFP+ group, and reached more than 75% by day 21. Examination of the normal CEM cells cultured with the conditioned media prepared from 50% GFP+ cells infected with HIV-1 revealed the appearance of GFP+ cells (Data not shown). This phenomenon was also observed when Sup-T1 and MT-2 cells were cultured with the cell-free conditioned medium. These data indicated the generation of replication-competent DLV in the HIV-1 infected transductant cultures, which confirmed the mobilzation of DLV.