Critical Factors Influencing Stable Transduction of Human CD34+ Cells with HIV-1-Derived Lentiviral Vectors

doi:10.1006/mthe.2000.0094, available online at http://www.idealibrary.com on IDEAL

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Critical Factors Influencing Stable Transduction of Human CD34+ Cells with HIV-1-Derived Lentiviral Vectors Dennis L. Haas,* Scott S. Case,† Gay M. Crooks,† and Donald B. Kohn*,†,1 *Department of Molecular Microbiology and Immunology, University of Southern California Medical School, Los Angeles, California 90033 †Division of Research Immunology/Bone Marrow Transplantation, Childrens Hospital Los Angeles, 4650 Sunset Boulevard Mailstop #62, Los Angeles, California 90027 Received for publication February 9, 2000, and accepted in revised form June 6, 2000

Lentiviral vectors have been proposed as a more efficient alternative to Moloney murine leukemia virus-based retroviral vectors for transduction of human hematopoietic progenitors and stem cells. These studies were designed to evaluate the conditions that influence transduction frequency of CD34+ progenitors, with the goal of optimizing efficiency of stable gene transfer with lentiviral vectors. CD34+ human cord blood cells and 293 cells were transduced with a human immunodeficiency virus (HIV)-1 derived lentiviral vector pseudotyped with vesicular stomatitis virus glycoprotein and carrying an internal human cytomegalovirus promoter driving enhanced green fluorescent protein (eGFP) expression. Using fluorescence-activated cell sorting analysis of eGFP, we observed pseudotransduction beginning at the time of vector addition and lasting up to 24 h in CD34+ cells and up to 72 h in 293 cells. Integrase-defective lentiviral vector caused transient eGFP expression for up to 10 days in CD34+ cells and for up to 14 days in 293 cells. Protamine sulfate conferred no increase in transduction efficiency of CD34+ cells on fibronectincoated plates. Transduction frequency was related directly to vector concentration and not to multiplicity of infection across the ranges tested. First- and second-generation lentiviral vectors transduced CD34+ cells equally, demonstrating a lack of dependence on HIV-1 accessory proteins. These findings will be useful for the optimal utilization of this new class of vectors for transduction of human hematopoietic stem cells. Key Words: lentiviral vector; CD34; transduction; hematopoietic stem cells; gene therapy.

INTRODUCTION Gene transfer into hematopoietic stem cells (HSC) may have important applications for the treatment of genetic diseases, cancer, and AIDS. However, despite more than a decade of studies, gene transfer into human HSC using Moloney murine leukemia virus (MLV)-based retroviral vectors has been found to be too inefficient, in most settings, to produce clinical benefit. This inefficient gene transfer is at least partly due to inherent characteristics of HSC; most HSC are in a quiescent state (1), are relatively slow to respond to cytokine stimulation (2–5), and when induced to divide tend to lose long-term repopulating capacity (6–11). Recently, new vectors have been developed from lentiviruses such as HIV-1. Lentiviral vectors may provide

1To whom correspondence should be addressed at Division of Research Immunology/Bone Marrow Transplantation, Childrens Hospital Los Angeles, 4650 Sunset Boulevard Mailstop #62, Los Angeles, CA 90027. Fax: 1-323-660-1904. E-mail: [email protected].

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the needed improvement in gene transfer efficiency because, unlike MLV-based retroviral vectors, they are capable of transducing nondividing cells, including neurons, hepatocytes, cardiac myocytes, and CD34+CD38− human hematopoietic cells (12–20). The nuclear localization signal present in the matrix protein of HIV-1 allows entry of the viral preintegration complex into the nucleus through the intact nuclear membrane (21), while MLV-based vectors require the nuclear membrane to break down during mitosis to gain access to the genome. This property may contribute to the lentiviral vector’s ability to transfer genes into nondividing HSC (20, 22). An additional contributor to inefficient gene transfer is the relative scarcity of MLV receptors on the surface of HSC, which may limit adsorbance of vector (23, 24). Pseudotyping lentivirus vectors with vesicular stomatitis virus glycoprotein (VSV-G) targets vector particles to membrane phospholipids of target cells (25), rather than relying on the expression of a specific receptor for binding. In addition, the VSV-G pseudotype produces a more stable vector particle that can withstand ultracentrifuga-

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ARTICLE tion, allowing for the concentration of vector supernatants to high titer (26). With the aim of defining conditions to achieve optimal transduction frequency of human CD34+ hematopoietic cells with this new class of vectors, we first demonstrated that two artifacts, pseudotransduction and transient expression of unintegrated vector, can complicate FACS analysis of transduction frequency by eGFP expression if cells are analyzed too soon after transduction. In addition, we showed that anchoring CD34+ cells with the fibronectin fragment CH-296, a common technique for increasing transduction frequency of CD34+ cells (27, 28), in the presence or absence of the polycation protamine sulfate, did not confer greater transduction frequency by VSV-G pseudotyped lentiviral vectors. We found that transduction efficiency was identical using first- or second-generation vectors, demonstrating that HIV-1 accessory proteins are not needed for stable transduction of CD34+ cells. Finally, we demonstrated that the transduction frequency of VSV-G pseudotyped lentiviral vectors is dependent on vector concentration and is independent of multiplicity of infection (m.o.i.), across the ranges tested. These findings should aid in the design of transduction conditions that achieve optimal gene transfer with this new vector system.

MATERIALS

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METHODS

Vector construction. The lentiviral vector plasmid pHR′CMVeGFP was constructed as previously described (16) and contains the enhanced green fluorescent protein (eGFP; Clontech Laboratories, Palo Alto, CA) reporter gene driven by the human cytomegalovirus (CMV) immediate-early promoter. The lentiviral vector plasmid pHR′CMVd4eGFP was constructed by replacing the eGFP gene from pHR′CMVeGFP with a destabilized form of eGFP having a fluorescence half-life of 4 h from pd4eGFP-N1 (Clontech Laboratories). The expression plasmid VR1012-CMVeGFP (Vical Inc., San Diego, CA) was used in place of vector plasmid to generate pseudo-lentiviral supernatants. The first-generation packaging plasmid pCMV∆R8.2 was used to express the HIV-1 Gag, Pol, Tat, Rev, Vif, Vpr, Vpu, and Nef proteins to package the lentiviral vectors without the HIV-1 Env protein (19). The second-generation packaging plasmid pCMV∆R8.91 was used to express the HIV-1 Gag, Pol, Tat, and Rev proteins to package the lentiviral vectors without the HIV-1 Env protein or the accessory proteins Vif, Vpu, Vpr, and Nef (19). The first-generation integrase-defective packaging plasmid pCMV∆R(int−)8.2, constructed as previously described (15), was used to generate integrase-defective LentiCMVeGFP/VSV lentiviral vector preparations. The envelope expression plasmid pMD.G(VSV) was used to express the vesicular stomatitis virus glycoprotein (VSV-G) from the human CMV immediate-early promoter (16). MLVeGFP vector preparations were generated with a gibbon ape leukemia virus (GALV) pseudotype from the stable packaging cell line PG13 (29). Supernatant production by transient three-plasmid transfection. VSV-G pseudotyped lentiviral vectors were produced by transient three-plasmid transfection of 293T cells (ATCC No. CRL-11268) as previously described (30) using 2 µg of envelope plasmid, 10 µg of packaging plasmid, and 10 µg of lentiviral vector plasmid. Twelve hours after transfection, sodium butyrate (Sigma Scientific, Inc., Brighton, MI) induction was performed by treating transfected cells with 10 mM sodium butyrate in D10 [Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mmol/liter L-glutamine] as previously described (30). After 12 h of exposure to sodium butyrate, the cells were washed twice with phosphate-buffered saline (PBS) and refed with fresh D10 medium. Thereafter, supernatants were

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collected every 12 h for 3–5 days, filtered through a 0.2-µm filter flask (Nalgene, Rochester, NY), and concentrated by ultracentrifugation at 50,000g for 140 min, as previously described (31). Pellets were resuspended in serum-free X-vivo 15 (BioWhittaker, Walkersville, MD) and stored at −80C until used. Supernatants titered by eGFP expression in 293 cells. Titers were determined by endpoint dilution. 293 cells (ATCC No. CRL-1573) were seeded at 1  105 cells/well in a six-well cell culture plate (Corning Inc., Miami, FL) in D10, placed in a 37C incubator for 12 h, and allowed to adhere. Cells were transduced with 1-ml serial dilutions (i.e., 10−1, 10−2, 10−3) of vector supernatants and analyzed by FACS for eGFP expression 48 h later. Titers were calculated using the following equation: (No. of 293 cells at the time of vector addition) (% eGFP(+)/100)/(1/dilution) = No. of infectious units (iu)/ml. Titers ranged between 0.5  106 and 10  106 iu/ml before ultracentrifugation and 1  108 and 15  108 iu/ml after ultracentrifugation. All lentiviral vector preparations were tested for the presence of replication-competent lentivirus (RCL) by infection of phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells, followed by culture for 2 weeks and then assay of culture medium for p24 gag by ELISA (Beckman Coulter Inc., Fullerton, CA). No vector preparations contained detectable RCL. Cell sources and isolation. For most experiments, CD34+ cells were isolated from human umbilical cord blood obtained from normal deliveries, using Miltenyi MiniMACS magnetic separation columns (Miltenyi Biotech, Sunnyvale, CA) after Ficoll–Hypaque (Amersham Pharmacia Biotech, Piscataway, NJ) density gradient centrifugation. Use of these cord blood samples was approved by the Committee on Clinical Investigations at Childrens Hospital, Los Angeles (32). CD34+ cells used for comparing transduction on fibronectin fragment CH-296 (Takara Shuzo Co., Otsu, Shiga, Japan) were further defined as cells with high CD34 expression alone or in some experiments cells with high CD34 and CD38 expression (CD34+CD38+) by FACS, as previously described (32). FACS analysis was performed on a FACSVantage [Becton–Dickinson Immunocytometry Systems (BDIS), San Jose, CA] using Lysys II software (BDIS). eGFP expression time courses. CD34+ cells were plated at a density of 7  104 cells/cm2 onto CH-296-coated, 35-mm plates in serum-free X-vivo 15 containing 5 ng/ml recombinant human interleukin (IL)-3 (Biosource International, Camarillo, CA), 16.5 U/ml IL-6 (Biosource International), and 25 ng/ml Steel Factor (SF) (Biosource International) and placed into a 5% CO2, 37C incubator. 293 cells were plated at a density of 1.2  106 cells/cm2 onto 35-mm plates in D10 and placed into a 5% CO2, 37C incubator. Two hours later, a single administration of concentrated lentiviral vector supernatant was added to produce a final concentration of 1  107 iu/ml in a final volume of 2 ml. Pseudo-lenti/VSV vector supernatants were packaged with pCMV∆R8.91, concentrated by ultracentrifugation, and used untitered. 293 cells were transduced in parallel with the same vector supernatants at a final concentration of 5  105 iu/ml in a final volume of 2 ml. At designated time points, 10–25% of the cells were harvested, fixed in 0.5% paraformaldehyde for 30 min at room temperature, and analyzed by FACS for eGFP expression. Transduction of CD34+ cells on CH-296-coated plates. CD34+ cells (1–10  104) were transduced in 2 ml of X-vivo 15 containing 5 ng/ml IL-3, 16.5 U/ml IL-6, and 25 ng/ml SF in 35-mm plates coated with either the recombinant fibronectin fragment CH-296 (Takara Shuzo Co.) or 2% bovine serum albumin (BSA) as a control. Transductions were performed with 1 addition of vector supernatant for 12 h immediately after CD34+ cell isolation. In some experiments, cells were transduced in medium containing 4 µg/ml protamine sulfate (Sigma–Aldrich, St. Louis, MO). LentiCMVeGFP/VSV transduction was performed with a vector concentration of 1  107 iu/ml. MLV/GALV transduction was performed with a vector concentration of 5  105 iu/ml. Mock (nontransduced) controls were handled exactly the same, but with no vector supernatants added to the CD34+ cells. Following transduction, CD34+ cells were cultured on irradiated allogeneic human bone marrow stroma in long-term bone marrow culture (LTBMC) medium (32). After 7 days, the cells were harvested with cell dissociation buffer, washed with PBS, fixed with 0.5% paraformaldehyde at room temperature, and analyzed for eGFP expression on a FACScan.

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FIG. 1. False eGFP positivity is visible by FACS analysis and is contributed by both pseudotransduction and transient expression from unintegrated vector. Representative FACS analysis to demonstrate the expression profile of pseudo-lenti/VSV, LentiCMVeGFP/VSV(int−), and LentiCMVeGFP/VSV transduced 293 cells. Diagonal gates were chosen based on untransduced mock populations to include cells with very low fluorescence intensity as positive events.

Transduction of CD34+ cells to examine the role of HIV-1 accessory proteins. CD34+ cells (1–10  104) were transduced in 2 ml of X-vivo 15 containing 5 ng/ml IL-3, 16.5 U/ml IL-6, and 25 ng/ml SF in 35-mm plates coated with either the recombinant fibronectin fragment CH-296 (Takara Shuzo Co.). Transductions were performed with one addition of vector supernatant for 12 h immediately after CD34+ cell isolation. Both firstand second-generation LentiCMVeGFP/VSV transductions were performed with a vector concentration of 1  107 iu/ml. Mock (nontransduced) controls were handled exactly the same, but with no vector supernatants added to the CD34+ cells. After 7 days, the cells were harvested with cell dissociation buffer, washed with PBS, fixed with 0.5% paraformaldehyde at room temperature, and analyzed for eGFP expression on a FACScan. Concentration vs m.o.i. transductions. For experiments that varied m.o.i. by altering vector supernatant volume, CD34+ cells were added to 16-mm cell culture plates (Corning Inc.) coated with the recombinant fibronectin fragment CH-296 (Takara Shuzo Co.), at a density of 2.1  103 cells/cm2 in serum-free X-vivo 15 containing 5 ng/ml IL-3, 16.5 U/ml IL6, and 25 ng/ml SF and placed into a 5% CO2, 37C incubator for 2 h to allow for cell adherence. For experiments that varied m.o.i. by altering target cell number, CD34+ cells were plated at 1.4  103, 4.2  103, and 1.3  104 cells/cm2. After 2 h, concentrated supernatant containing LentiCMVeGFP/VSV vector packaged with pCMV∆R8.91 was added to the cells in specific volumes of medium needed to make the appropriate final concentration and m.o.i. and the cells were placed back into the 5% CO2, 37C incubator. After 12 h of exposure to the vector, the cells were harvested with cell dissociation buffer (GibcoBRL, Rockville, MD), washed two times with PBS, and spun at 500g for 5–7 min between washes. The cells were then placed onto fresh CH-296-coated 16-mm plates in basal bone marrow medium [BBMM; Iscove’s modified Dulbecco’s medium (IMDM) with 30% FCS, 1% BSA (Sigma Scientific, Inc.), 10−4 mol/liter 2mercaptoethanol, 10−6 mol/liter hydrocortisone, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mmol/liter L-glutamine) containing the aforementioned cytokine cocktail and cultured in a 5% CO2, 37C incubator for 5 days. After 5 days, the cells were harvested with cell dissociation buffer, washed two times with PBS, fixed with 0.5% paraformaldehyde for 30 min at room temperature, and analyzed for eGFP expression on a FACScan (BDIS).

MOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright  The American Society of Gene Therapy

RESULTS False eGFP Positivity Is Visible by FACS Analysis and Is Contributed by Both Pseudotransduction and Transient Expression from Unintegrated Vector To determine how soon after vector administration eGFP positivity by FACS can be used as a reliable indicator of stable transduction, we established the time course of eGFP positivity in transduced CD34+ cells. We also examined the time course of eGFP positivity in the human embryonic kidney cell line 293 because they are permissive to lentiviral vector transduction and were used to determine vector titers in this study. False eGFP positivity at early times after vector administration can be caused by at least two mechanisms. The first mechanism is pseudotransduction, or the direct transfer of marker gene protein by either its presence in vector supernatants or its incidental incorporation into the vector particle (31). The second mechanism is transient expression of the vector’s marker gene inside a target cell, prior to the vector’s integration into that target cell’s genome. To determine if pseudotransduction is possible under our vector production and target cell transduction conditions, we generated pseudo-lentiviral vector supernatants using the same three-plasmid transfection protocol used to generate lentiviral vector supernatants, except the lentiviral vector plasmid was substituted with a human CMV promoter-driven eGFP expression plasmid. Under these conditions eGFP protein is still produced by 293T packaging cells, but no vector transcript is available for incorporation into vector particles. These pseudo-lentivi-

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FIG. 2. Time course of false eGFP positivity contributed by pseudotransduction and transient expression from unintegrated vector. eGFP positivity by FACS analysis of 293 cells and CD34+ cells after a single administration of “pseudo”-lenti/VSV, LentiCMVeGFP/VSV(int−), or LentiCMVeGFP/VSV supernatants.

ral vector supernatants would contain free eGFP protein and could contain vector particles devoid of vector genome, but containing eGFP protein. Figure 1 shows the FACS expression profile of 293 cells 12 h after a single administration of pseudo-lentiviral vector supernatant. At this early time point, there was a clearly detectable population of cells with low fluorescence intensity. These low-intensity eGFP-positive cells were no longer visible by FACS when analyzed 7 days after vector administration. To determine how long cells remain eGFP positive due to pseudotransduction, we established the time course of eGFP positivity in both 293 cells and CD34+ cells (Fig. 2) after a single administration of pseudolentiviral vector supernatant. Figure 2 demonstrates that pseudotransduction occurred in both cell types and was visible by FACS analysis beginning immediately after pseudo-lentiviral vector supernatant addition and reached a maximum at, or before, the 12-h time point. Very-low-level eGFP positivity was still visible at 72 h in 293 cells (Fig. 2A) and 24 h in CD34+ cells (Fig. 2B). Although cell-type-specific differences in the frequency of pseudotransduction were consistently observed among multiple experiments, differences in the frequency of pseudotransduction among the three different vector supernatant preparations within each experiment were not. The differences in the frequency of pseudotransduction among the three different vector supernatant preparations observed in Fig. 2 are believed to reflect variability in the level of pseudotransduction contributed by different vector supernatant preparations and

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not due to vector-specific differences. In other words, different vector supernatant preparations of the same vector were observed to contribute variable frequencies of pseudotransduction between experiments. To examine whether transient expression from unintegrated vector is possible under our vector production and target cell transduction conditions, we generated integrasedefective lentiviral vector supernatants. We generated these integrase-defective vector supernatants using the same three-plasmid transfection protocol used to generate viable lentiviral vector supernatants, except the packaging plasmid was substituted with one coding for a nonfunctional integrase protein (15). Under these conditions, supernatants would be expected to contain vector particles, devoid of functional integrase protein, and so be unable to integrate into the target cell genome. Figure 1 shows the FACS expression profile of eGFP-positive cells 12 h after a single administration of integrase-defective lentiviral vector supernatant. The dimly positive events at this early time point after transduction with pseudo-lentiviral vector, integrase-defective lentiviral vector, and complete, integrasecompetent lentiviral vector appeared very similar, suggesting that in each case these events were probably due to pseudotransduction. In contrast to cells transduced with pseudo-lentiviral vector supernatants, a small but significant fraction of cells transduced with the integrase-defective vector were still eGFP positive at 7 days after transduction. Some of these cells showed high eGFP fluorescence intensity, suggesting that vector integration is not a requirement for expression or that a small percentage of integration occurs by a non-integrase-mediated mechanism.

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FIG. 3. Integrase-defective lentivirus vectors expressing destabilized eGFP appear positive for a shorter duration. To test whether the extended eGFP positivity of cells transduced with integrase-defective vector was due in part to the stability of eGFP, we constructed an analogous LentiCMVeGFP/VSV vector expressing a destabilized form of eGFP having a fluorescence half-life of 4 h, called LentiCMVd4eGFP/VSV. A shows the time course of eGFP positivity of 293 cells. B shows a representative FACS analysis to demonstrate the difference in MFI at 48 h after transduction.

To determine how long cells remained positive due to transient expression of unintegrated vector, we established the time course of eGFP positivity in both 293 cells and CD34+ cells (Fig. 2) after a single administration of integrase-defective lentiviral vector supernatant. Figure 2 demonstrates that transient expression was still visible by FACS analysis at 14 days after transduction in 293 cells (Fig. 2A) and 10 days in CD34+ cells (Fig. 2B). These findings suggest that the same expression cassette contained within the complete integrase-competent lentiviral vector is presumably capable of expressing eGFP prior to its integration into the target cell genome. One reason for extended eGFP positivity of cells transduced with integrase-defective vector may be the stability of eGFP, which usually has a fluorescence halflife of greater than 24 h (33). To test whether extended eGFP positivity of cells transduced with integrase-defective vector was due to the stability of eGFP, we constructed an analogous LentiCMVeGFP/VSV vector expressing a destabilized form of eGFP having a fluorescence half-life of 4 h (34) called LentiCMVd4eGFP/VSV. Figure 3A demonstrates that 293 cells transduced with integrase-defective vector expressing the destabilized, 4-h half-life eGFP remained positive by FACS only until day 4, while integrase-defective vector expressing the 24-h half-life protein remained positive until day 14. This finding demonstrates that much of the duration of eGFP false positivity contributed by unintegrated vector is due to the stability of eGFP that is expressed before day 4 after transduction. In addition, Fig. 3A shows that the transduction efficiency of the lentiviral vector expressing the 24-h half-life eGFP appears to be greater than the MOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright  The American Society of Gene Therapy

lentiviral vector expressing the 4-h half-life eGFP. Figure 3B shows sample FACS to demonstrate that cells transduced with the destabilized eGFP had a lower mean fluorescence intensity (MFI) compared to cells transduced with the 24-h half-life eGFP. The lower MFI is presumably due to lower accumulation of eGFP in transduced cells. It is likely that the lower MFI contributes to at least some of the apparent loss in transduction efficiency from the vector expressing destabilized eGFP. Taken together, these results demonstrate that care must be taken when interpreting eGFP expression data gathered by FACS analysis at early time points after transduction.

Fibronectin Fragment CH-296 Does Not Increase Transduction Efficiency of CD34+ Cells by VSV-G Pseudotyped Lentiviral Vectors To determine if the recombinant fibronectin fragment CH-296 could increase transduction frequency of CD34+ cells with VSV-G pseudotyped lentiviral vectors, we compared the transduction of CD34+ cells by either LentiCMVeGFP/VSV or MLV/GALV by FACS analysis of eGFP expression. Figure 4 demonstrates that LentiCMVeGFP/VSV does not require the CH-296 fragment to transduce CD34+ cells as the transduction efficiency was not significantly different than cells transduced in suspension on BSA-coated control plates. At 7 days after transduction, eGFP expressions with LentiCMVeGFP/VSV were 22.3 ± 3.5% (n = 9) on CH-296 and 19.3 ± 1.4% (n = 7) (P = 0.22 for CH-296 compared

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FIG. 4. Fibronectin fragment CH-296 does not increase transduction efficiency of cord blood CD34+ cells by VSV-G pseudotyped lentiviral vectors in the presence or absence of protamine sulfate. To determine if the recombinant fibronectin fragment CH-296 (A) could increase transduction frequency of CD34+ cells in the presence or absence of protamine sulfate (B), with VSV-G pseudotyped lentiviral vectors, we compared the transduction of CD34+ cells by either LentiCMVeGFP/VSV or MLV/GALV (A), by day 7 FACS analysis of eGFP expression.

to BSA-coated plates). As has been previously reported, transduction with MLVeGFP/GALV was significantly increased in the presence of CH-296 compared to BSAcoated control plates (35). Figure 4B demonstrates that addition of the polycation, protamine sulfate, to the transduction medium at a concentration of 4 µg/ml did not affect the transduction efficiency of CD34+ with lentiviral vectors pseudotyped with VSV-G. It has previously been shown that VSV-G pseudotyped vectors require 10- to 100-fold higher concentrations of vector to achieve levels of transduction comparable to GALV pseudotyped vectors (36). This explains the higher relative transduction frequency by the GALV pseudotype MLV vector observed in Fig. 4.

of vector particles in the transduction medium or by increasing the total number of vector particles available to each cell (i.e., m.o.i.) in the transduction medium. Previous studies examining transduction by retroviral vectors pseudotyped with envelope glycoproteins that bind specific cellular receptors have demonstrated that transduction frequency is dependent on vector supernatant titer (37) and independent of target cell number (38). m.o.i. is a ratio of the total number of vector parti-

HIV-1 Accessory Proteins Do Not Affect Transduction Efficiency of CD34+ Cells by VSV-G Pseudotyped Lentiviral Vectors To determine if the accessory proteins of HIV-1 play a significant role in the transduction of CD34+ cells, we compared the transduction efficiency of these cells by first- and second-generation lentiviral vectors. Figure 5 demonstrates that both transduction efficiency and short-term expression stability were identical for both generations of lentiviral vectors.

Transduction of CD34+ Cells by VSV-G Pseudotyped Lentiviral Vectors Is Related Directly to Vector Concentration and Not to Multiplicity of Infection We determined whether the frequency of gene transfer would be augmented by increasing the concentration

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FIG. 5. HIV-1 accessory proteins do not affect transduction efficiency of CD34+ cells by VSV-G pseudotyped lentiviral vectors. To determine if the accessory proteins of HIV-1 play a significant role in the transduction of CD34+ cells, we compared the transduction efficiency of these cells by first- and second-generation vectors. First-generation vectors were packaged in the presence of the four HIV-1 accessory proteins Vif, Vpr, Vpu, and Nef. Second-generation vectors were packaged without these accessory proteins. The figure demonstrates that transduction efficiency is identical for both generations of lentiviral vectors.

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FIG. 6. Transduction of CD34+ cells by VSV-G pseudotyped lentiviral vectors is related directly to vector concentration and not to multiplicity of infection. The level of gene transfer into human CD34+ cells by the VSV-G pseudotyped lentiviral vector is increased by increasing the concentration of vector particles in the transduction medium, but not by increasing the multiplicity of infection. By adjusting vector supernatant volume (A and B) or target cell number (C and D), transductions performed with each concentration of vector tested using three different m.o.i.’s. Similarly, transductions performed with each m.o.i. were tested using three different concentrations of vector. A and C show the percentage of eGFP(+) cells vs vector concentration, while B shows the same data from A

cles divided by the total number of target cells. By adjusting vector supernatant volumes, transductions were performed in series so that each transduction was part of a serial dilution of vector concentration with m.o.i. held constant (Fig. 6A) and simultaneously part of a serial dilution of m.o.i. with vector concentration held MOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright  The American Society of Gene Therapy

constant (Fig. 6B). Similarly, by adjusting target cell number, transductions were performed in series so that each transduction was part of a serial dilution of vector concentration with m.o.i. held constant (Fig. 6C) and simultaneously part of a serial dilution of m.o.i. with vector concentration held constant (Fig. 6D).

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FIG. 7. Maximum vector concentration leading to increased gene transfer. Since the level of gene transfer is related directly to the concentration of LentiCMVeGFP/VSV particles in the transduction medium and not to the total number of vector particles available to each cell (m.o.i.), the data from three experiments similar to that shown in Fig. 6 were compiled and plotted against concentration, irregardless of m.o.i., to determine the maximum concentration that leads to an increase in gene transfer. The inset shows the same data plotted as % eGFP(+) vs % eGFP(+)/vector concentration (Eadie–Hofstee plot).

Figures 6A and 6C show the percentage of eGFP-positive cells determined by FACS analysis 5 days after transduction, plotted versus vector concentration, while Figs. 6B and 6D show the same data plotted versus m.o.i. These results indicate that increasing m.o.i. by either increasing vector supernatant volume or decreasing target cell number, while keeping vector concentration constant, did not lead to an increase in the percentage of eGFP-positive cells. However, increasing vector concentration, while keeping m.o.i. constant, did lead to an increase in the percentage of eGFP cells. Taken together, these results indicate that within the ranges tested, the extent of gene transfer can be increased by increasing the concentration of LentiCMVeGFP/VSV vector particles in the transduction medium, but not by increasing the m.o.i. by varying vector supernatant volume or target cell number. Based on the results from Fig. 6, we determined the maximum concentration that leads to an increase in gene transfer under these transduction conditions. Since the efficiency of gene transfer was directly related to the concentration of lentiviral vector particles in the transduction medium and not to the total number of vector particles available to each cell, data from three experiments similar to that shown in Fig. 6 were compiled and plotted against concentration, regardless of m.o.i. Vector concentrations ranged from 3.86  103 to 1.80  108 iu/ml. Plotting these data as concentration versus percentage of eGFP(+) cells (Fig. 7) reveals an exponential growth curve approaching a maximum. Data were plotted using Sigma Plot (SPSS, Inc., Chicago, IL) and a regression equation was obtained for an exponential growth curve with a maximum. The inset of Fig. 7 shows

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the same data plotted as % eGFP(+) vs % eGFP(+)/vector concentration (Eadie–Hofstee plot). Using this type of plot, the x-intercept indicates the maximum percentage of gene transfer that can be obtained under these transduction conditions. Using this number in the regression equation derived from the data in Fig. 7 and solving for concentration gives a concentration maximum of approximately 1.0  108 iu/ml, beyond which a further increase in gene transfer under these transduction conditions cannot be expected, solely by increasing vector concentration.

DISCUSSION Lentiviral vectors derived from HIV-1 show great promise for the transduction of nondividing cells such as hematopoietic stem cells. As the use of transduction conditions previously established using the MLV-based vector system may not translate into the optimal use of this new vector system, the critical factors influencing gene transfer by VSV-G pseudotyped lentiviral vectors into CD34+ cells were explored. The frequency of eGFP positivity which we observed to be contributed by both pseudotransduction and transient expression from unintegrated vector demonstrates that care must be taken when interpreting eGFP as a measure of gene transfer, especially at times soon after transduction with VSV-G pseudotyped lentiviral vectors. Our established titering assay analyzes eGFP expression on 293 cells at 48 h after vector supernatant addition. The eGFP positivity time course in Fig. 2 establishes that this time point includes a contribution of false eGFP positivity by both pseudotransduction and potenMOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright  The American Society of Gene Therapy

ARTICLE tially transient expression from vector that subsequently fails to integrate (i.e., an integration-competent vector that is unintegrated and expressing at 48 h may or may not integrate at a later time). In addition, the time course demonstrates that at 48 h not all cells that have internalized fully competent vector are expressing eGFP at sufficient levels to be positive by FACS. As all functional titering assays are relative measures of viable vector or virus, and because each of our titering assays was performed in the same way, the fact that our titers include false positives and do not include all true positives is not important in establishing the basic phenomena illustrated in these studies. In addition, the nonstandardization of titer measurements indicates that the concentration yielding maximum transduction reported here may be different in a different experimental system or if the vector supernatants are titered using a different assay. Fibronectin has been shown to increase transduction frequency by both amphotropic and GALV pseudotyped retroviral vectors, presumably due to colocalization through simultaneous binding of both the target cell and vector (28, 35). Therefore, the fact that CH-296 does not increase transduction frequency by lentiviral vectors pseudotyped with VSV-G is presumably due to the inability of VSV-G to simultaneously bind the fibronectin fragment and the target cell. Alternatively, localization by fibronectin may occur, but as VSV-G does not rely on the expression level of specific receptors, target cell vector contact may not be a limiting factor for transduction. Though fibronectin does not increase transduction efficiency with VSV-G pseudotyped vectors, we continue to use it in our transduction studies because it has been shown to have growth-supporting activity both in vitro and in vivo (39) and to maintain the multilineage regenerative capacity of primitive hematopoietic cells that have traversed M phase during ex vivo culture (40). In our study, addition of the polycation protamine sulfate to transduction medium also did not increase transduction frequency. It has previously been shown that protamine sulfate enhanced the attachment of VSV to HeLa cells, but reduced VSV attachment to avian CER cells, suggesting that although electrostatic interactions play a role in the binding of VSV to the cell membrane, more specific structural features are required for viral attachment (41). We conclude that vector concentration has a greater influence on gene transfer efficiency than multiplicity of infection, in the ranges tested in this study. This means that using and reporting vector concentration is more accurate and informative than using and reporting m.o.i. Transduction experiments performed using multiplicity of infection as the guiding parameter may be designed in an unlimited number of ways by using different cell numbers and different volumes of vector supernatants of different concentrations. Variability in experimental design can result in suboptimal transduction efficiency and inconsistent reproducibility between experiments. Although not surprising, the observation that transduction efficiency is directly related to vector concentration, MOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright  The American Society of Gene Therapy

rather than to multiplicity of infection, is important for establishing basic transduction conditions and reproducibility among experiments and among investigators. We determined the maximum concentration that leads to further gene transfer into CD34+ cells under these transduction conditions to be approximately 1.0 108 iu/ml. It is possible that this maximum value is underestimated due to the lack of data points using vector supernatant concentrations higher than those we can currently generate by transient transfection and ultracentrifugation. We have observed that the maximum gene transfer in a single-administration, 12-h transduction using high-concentration supernatants can be overcome using multiple vector administrations. This suggests that the transduction maximum is limited by the frequency of cells that are available for transduction at the time of vector addition. Although lentiviral vectors are capable of transducing nondividing cells (20), it has been shown that transduction occurs at different levels depending on cell cycle state of the target cell (42). Taken together, these results demonstrate that the conditions for optimal transduction of CD34+ hematopoietic cells should include a high vector concentration of at least 1  107 iu/ml. Since transduction efficiency is not related to m.o.i., it is not necessary, or economical, to transduce cells in large volumes of supernatant. Even though maximum transduction frequency was shown to occur near a vector concentration of 108 iu/ml, we routinely use concentrations in the range of 107 iu/ml because this concentration still results in a large fraction of maximal transduction, but consumes considerably less vector. In addition, analysis by eGFP marker gene expression alone should not be considered an accurate measure of stable gene transfer unless cells are analyzed 4 days after transduction (36). At this time, pseudotransduction is no longer detectable, and although transient expression of vector is still possible, the eGFP expression time course data suggest that eGFP expression remains stable after day 4. The findings presented here will help in further defining the optimal parameters for maximal transduction of pluripotent HSC. Ongoing studies analyzing gene transfer into long-term repopulating cells of large animals and xenograft models to study human long-term repopulating cells will provide further information on the potential that lentiviruses offer for HSC transduction in a clinical setting. ACKNOWLEDGMENTS We thank Inder M. Verma and Luigi Naldini for helpful discussion throughout this project and Lora Barsky and David Bockstoce for technical assistance with flow cytometry. This work was supported by National Institutes of Health Grants RO1DK54567, RO1DK56287, 1P50HL54850, and 5P01CA59318 and a Leukemia Society of America Translational Research Award (G.M.C.) D.L.H. is the recipient of National Institutes of Health/National Cancer Institute, Program in Molecular Oncology Training Grant 2 T32 CAO9569-11Al. S.S.C. is the recipient of a fellowship from the Childrens Hospital Los Angeles Research Institute. D.B.K. is the recipient of an Elizabeth Glaser Scientist Award from the Pediatric AIDS Foundation. G.M.C. is a Leukemia Society of America Scholar.

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