Centrifugal enhancement of retroviral mediated gene transfer

ELSEVIER

Journal of Virological Methods Journal of Virological Methods 54 (199.5) 131-143

Centrifugal enhancement of retroviral mediated gene transfer Alfred B. Bahnson aY *, James T. Dunigan a, Bora E. Baysal a, Trina Mohney a, R. Wayne Atchison b, Maya T. Nimgaonkar ‘, Edward D. Ball ‘, John A. Barranger a a Department

of Human Genetrcs, Graduate School of Pubhc Health, University of Pittsburgh,

Pittsburgh. PA 15261, USA b Magee Womens Research Institute, University of Pittsburgh Medical Center, Pittsburgh, PA, USA ’ Diviszon of Hematology/ Bone Marrow Transplantation, Unruersity of Pittsburgh Medical Center, Pittsburgh, PA, USA

Accepted 3 April 1995

Abstract Centrifugation has been used for many years to enhance infection of cultured cells with a variety of different types of viruses, but it has only recently been demonstrated to be effective for retroviruses (Ho et al. (1993) J. Leukocyte Biol. 53, 208-212; Kotani et al. (1994) Hum. Gene Ther. 5, 19-28). Centrifugation was investigated as a means of increasing the transduction of a retroviral vector for gene transfer into cells with the potential for transplantation and engraftment in human patients suffering from genetic disease, i.e., gene therapy. It was found that centrifugation significantly increased the rate of transduction into adherent murine fibroblasts and into non-adherent human hematopoietic cells, including primary CD34 + enriched cells. The latter samples include cells capable of reconstitution of hematopoiesis in myeloablated patients. As a step toward optimization of this method, it was shown that effective transduction is: (1) achieved at room temperature; (2) directly related to time of centrifugation and to relative centrifugal force up to 10,000 g; (3) independent of volume of supernatant for volumes > 0.5 ml using non-adherent cell targets in test tubes, but dependent upon volume for coverage of adherent cell targets in flat bottom plates; and (4) inversely related to cell numbers per tube using non-adherent cells. The results support the proposal that centrifugation increases the reversible binding of virus

* Corresponding author. Fax: + 1 (412) 383 9760. 0166-0934/9.5/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDIO166-0934(95)00035-6

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to the cells, and together with results reported by Hodgkin et al. (Hodgkin et al. (1988) J. Viral. Methods 22, 215-230), these data support a model in which the centrifugal field counteracts

forces of diffusion which lead to dissociation during the reversible phase of binding. Keywords:

Centrifugal enhancement; Retroviral gene transfer, Transduction efficiency; Gene therapy

1. Introduction Applications of viral mediated gene transfer now include ex vivo transduction of human cells for clinical purposes, i.e., gene therapy and gene marking studies. A problem with some protocols is low or inconsistent transduction efficiency (Dunbar et al., 1993; Kohn et al., 1993; von Kalle et al., 1993; Bahnson et al., 1994; Deisseroth et al., 1994; Hoogerbrugge et al., 1994; Nimgaonkar et al., 1994). Methods to enhance the transduction process could have broad applicability. For retroviral vectors, polybrene or protamine sulfate is universally used to enhance infection. Other methods of increasing transduction efficiency have focused on increasing the virus titer by generation and selection of high-titer producer cell clones, by control of conditions for virus production, and by concentration of virus-containing supernatants. It has long been known that centrifugation can enhance viral infection of cells. This technique is used in screening clinical specimens for a variety of pathological viruses and other intracellular parasites (see references in Hodgkin et al., 19881, including the human immunodeficiency virus (Ho et al., 1993). Centrifugation was tested as a means to enhance transduction of a murine retroviral vector (MFG-GC) carrying the human glucocerebrosidase gene. The aim of these studies was two-fold: (1) to improve the efficiency of transduction for generating and screening producer cells; and (2) to improve the transduction efficiency of human hematopoietic cells for clinical trials of gene therapy for Gaucher disease. This study shows that centrifugation significantly enhances retroviral transduction of adherent murine fibroblasts (NIH 3T31, human erythroleukemia cells (TF-I), and primary human CD34 + enriched hematopoietic cells. The effect of centrifugal force, centrifugation time, cell number, and other variables was examined on transduction efficiencies. The results confirm the usefulness of centrifugation for enhancing retroviral transduction of cells in culture and provide a basis for further optimization of conditions. A model for illustrating the forces involved in centrifugal enhancement is described.

2. Materials

and methods

2.1. Vector-containing

supernatant

Retroviral vector-containing supernatant (supe) was collected from confluent monolayers of SCRIP packaging cells (Danos and Mulligan, 1988) grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco/BRL) with 10% calf serum (CS; Gibco/ BRL) producing an MFG-GC vector containing the human cDNA for glucocerebrosidase (Ohashi et al., 1992). The titer of the full strength supe was approximately 1 X lo7

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infectious units/ml based upon infection (Bahnson et al., 1994). Supes were filtered In all infection experiments, control cells diluted in appropriate culture medium and sulfate (Sigma). 2.2. Transduction

133

of NIH 3T3 cells under standard conditions (0.45 pm) and stored at - 70°C prior to use. were mock-transduced with lO%CS/DMEM containing polycation, polybrene or protamine

of NIH 3T3 cells

NIH 3T3 cells were seeded at 5 X lo4 cells/well in 6-well plates 1 day prior to infection. At the time of infection, medium was removed by aspiration and replaced with mixtures containing supe diluted l/2 or more in fresh medium (lO%CS/DMEM) and polybrene (8 pg/ml). Centrifugation was performed in microtiter plate carriers using a Sorvall 6000B table-top centrifuge. After the infection period, the supe mixture was replaced with fresh medium, and the plates were incubated under normal conditions (5% CO,, 100% humidity, 37°C) for 2 days prior to harvest. Centrifugation of 3T3 targets in 6-well plates for 1 h using 3 ml/well diluted supernatants containing 12.5 mM HEPES at 2000 g was determined to be effective for routine use. 2.3. Transduction

of TF-1 cells

TF-1 non-adherent factor-dependent human erythroleukemia cells (American Type Culture Collection), maintained in log-phase growth in RPM1 medium (Gibco/BRL) containing 10% fetal bovine serum and 5 ng/ml GM-CSF (Immunex Research and Development Corporation), were suspended in mixtures of supe and fresh medium containing protamine sulfate (4 pg/ml) and were distributed at indicated cell numbers per tube in various types of test tubes. Fifteen-milliliter polystyrene culture tubes (17 X 100 mm; Fisher) and 15-ml polypropylene centrifuge tubes (Sarstedt) were centrifuged in a Sorvall 6000B table-top refrigerated centrifuge, microcentrifuge tubes were centrifuged in an Eppendorf 5415C variable speed microcentrifuge, and cryotubes were centrifuged either in the microcentrifuge or in a Sorvall RCSC refrigerated high speed centrifuge by positioning and supporting the tubes with crumpled paper in the wells of a fixed angle rotor. After the period of infection, the supe mixture was removed and cells were suspended in fresh culture medium and were transferred to 6- or 24-well plates (Falcon) for incubation under normal conditions. 2.4. Transduction

of cord blood CD34 +

cells

Cord blood CD34 + cells were enriched from mononucleated cells using immunoaffinity columns (CellPro, Inc.), and were prestimulated for 1 day in long-term bone marrow culture medium (LTMC) containing 10 ng/ml Interleukin (IL)-3, IL-6, and stem cell factor (SCF) (Immunex Research and Development Corporation). LTMC consisted of 12.5% fetal bovine serum (FBS; Hyclone Laboratories), 12.5% horse serum (HS; Hyclone Laboratories), 1 PM hydrocortisone succinate, 10 PM a-thioglycerol, and 1 PM 2-mercaptoethanol in Iscove’s modified Dulbecco’s medium (IMDM; Gibco/BRL). Between 5 X lo4 and 3 X lo5 cells were added per 15-ml culture tube

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with 1 ml of supe mixture (l/2 dilution) containing protamine sulfate (4 pg/ml). The tubes were incubated briefly for CO, equilibration and then were centrifuged for 2 h at 2400 g at 20°C in the Sorvall 6000B table-top centrifuge. Between centrifugations, the cells were incubated without replacement of medium and without transfer from the culture tubes. Centrifugation was repeated with replacement of l/2 of the medium with fresh supe (plus cytokines and protamine sulfate) two additional times over 2 days. Transduced cells and control non-transduced cells were expanded in LTMC with IL-3, IL-6, and SCF (10 ng/ml each) for up to 3 weeks prior to harvest and analysis for GC enzyme activity. For both TF-1 and CD34 + enriched cells, we typically transduce 3 X lo5 cells per tube with 1 ml of diluted supernatant in 15-ml round-bottom culture tubes for 2 h at 2000-2400 g at room temperature in a swinging bucket rotor. 2.5. Glucocerebrosidase

(CC) enzyme assay

Enzyme activity was determined using the fluorogenic substrate, 4-methylumbelliferyl glucopyranoside (Sigma), and sonicated cell lysates from cells harvested at least 48 h after infection. Specific GC activity is expressed in units (nmol fluorescent product converted per hour) per milligram of protein (Bahnson et al., 1994). Cell concentration and viability were determined using a hemocytometer and eosin exclusion.

3. Results 3.1. Transduction

of murine 3T3 embryonic fibroblasts

It was previously shown, using the MFG-GC vector, that integrated vector copy numbers up to 60 copies per genome correlate linearly with elevation in specific enzyme expression in non-clonal 3T3 targets at GC specific activities up to 4000 U/mg (Bahnson et al., 1994). For the purposes of this study, therefore, glucocerebrosidase enzyme expression in target cells within this range as a measure of transduction efficiency was relied upon. Experiments were initially conducted with adherent 3T3 cells plated into 6-well plates, comparing non-centrifuged to centrifuged in microtiter plate carriers at room temperature and at 34°C (Fig. 1). The centrifuge temperature was set below 37°C to reduce the possibility that an inaccurate setting could lead to overheating. Temperature had no effect on centrifugal enhancement, and the dose-response curve for dilutions of supernatant ranging from 0.02 to 0.5 demonstrates that an exponential relationship between transduction efficiency and virus concentration governs the infection process with centrifugation, similar to results obtained under normal gravitational forces (Bahnson et al., 1994). The elevation of enzyme activity above background levels (endogenous enzyme activity was approximately 210 U/mg for 3T3 cells) in centrifuged samples was over 20-fold that of non-centrifuged samples at the highest supe dilution (0.02) and between 3- and lo-fold higher for more concentrated supes. For this experiment, the supe was replaced with fresh medium without rinsing after 1 h exposure, and it is likely

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-m- 1sooxg; 22 c -m-ISOOxg; +-

0

0.2

0.1

Concentration

0.3

of Supematant

34 c

lxg; 37 C incubator

0.4

0.6

Mixture

Fig. 1. Centrifugal enhancement is effective at room temperature. Various dilutions of supe mixed with fresh medium (3 ml containing 12.5 mM HEPES) were added to NIH 3T3 cells in 6-well plates and centrifugation was performed in a refrigerated Sorvall 6000B set to 34 or 22°C for 1.5 h. Heat from the motor raised the temperature to the set point. Non-centrifuged control samples were incubated during this period at 37°C. Following the exposure period, the supe mixtures were replaced with fresh medium.

that residual at 1 g. Following within each plate carrier plates tend Nevertheless, enhancement

virus in the more concentrated

samples augmented

the observed expression

centrifugation, bare spots were observed in the lower/inner quadrant well. As an explanation, the position of the center of gravity of the loaded may differ from the position of the medium in the wells, and the flat-bottom to pool medium towards the outside where the radius is slightly greater. these results provided convincing evidence for a significant centrifugal of transduction during the l-h infection period.

3.2. Transduction of non-adherent

TF-1 human erythroleukemia

cells

For transduction of attached fibroblasts, the above results show that centrifugal enhancement can provide more efficient and more rapid transduction than non-centrifugal methods. However, we wanted to investigate the effect of centrifugation on non-adherent human hematopoietic stem cells. The factor-dependent human erythroleukemia cell line, TF-1 (Kitamura et al., 1989) was chosen as a model for preliminary studies to determine conditions for optimal enhancement. To determine whether supernatant volume had any effect on transduction efficiency in the test tube, without the complication of dead spots which had occurred with 6-well plates, 3 X 10’ TF-1 cells were aliquotted per tube, and volumes of OS-12 ml diluted supematant mixture were added. The tubes were centrifuged at 1500 g for 1 h. The results indicated that volume, and hence total virus number per tube, had no effect on the transduction level. This result also indicated that the effect of centrifugation is not due to increased pressure, which is dependent upon the height of the fluid column and upon the centrifugal field, and which would be more than lo-fold greater at the bottom of a tube containing 12 ml in comparison to 1 ml of fluid. However, there was a

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v Microcentrifuge

Tubes _ -,

0

2000

4000

6000

Relative Centrifugal

3000

10000

12000

Force (xg)

Fig. 2. Comparison of transduction in round bottom and conical bottom tubes at different field strengths. Aliquots of 1 ml of 3 X 10’ TF-1 cells/ml in a 1: 4 supe mixture were centrifuged in 2-ml round-bottom cryotubes and in 1.5-ml conical-bottom microcentrifuge tubes at various speeds for 1 h in an Eppendorf 5415C adjustable speed microcentrifuge.

difference between transduction in round-bottom tubes compared with conical-bottom tubes (mean + S.E.M. = 92 k 4 vs 80 k 1 U/mg, c-test: P < 0.05), suggesting that the more gentle radius of curvature of the round-bottom tubes improved exposure of the cells to the virus-containing medium. A similar difference was observed between infection in cryotubes (round bottom) and microcentrifuge tubes (conical bottom) when samples of a suspension of TF-1 cells in a virus-containing supe mixture were centrifuged at differing speeds in a microcentrifuge (Fig. 2). Further results describing the dependence of transduction upon relative centrifugal force are described below. The difference observed between transduction in different shaped tubes implied that cell crowding might be an important variable. Therefore, the effect of varying cell numbers was examined using 15-ml round-bottom culture tubes with a constant volume of supernatant mixture. A pronounced effect was observed. Transduction efficiency declined exponentially with increasing cell numbers, such that for cell numbers over 1 X 106, there was no elevation above control levels of GC expression (Fig. 3, lower curve). To achieve more highly transducing conditions, cells and supematant mixtures were centrifuged in 2-ml cryotubes, and as a control measure, a comparison was made between centrifugation in the microcentrifuge and the thermally controlled (20°C) high-speed centrifuge. There was no difference between the average results obtained at 5000 g with the microcentrifuge in comparison to the high-speed centrifuge, although the precision appeared to be better with the latter centrifuge (Fig. 3, upper curve). These results confirmed the importance of cell numbers on transduction efficiency. Each log decrease in cell number resulted in an approximate two-fold increase in transduction efficiency. The regression line fitted to these log transformed data displays a slope, m, nearly identical to that obtained for samples expressing above-background levels of vector product in the experiment at 1500 g (m = -0.368 vs - 0.365, respectively, where y = Cx” and where y = enzyme activity (U/mg), x = cell number and C = a constant).

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Microfuge

l

High-speed

-y

100,000

- 5000xg - 5000xg

= c XA(Al.37)

-a- -

10,000

137

Tabletop - 15OOxg Noninfected

1,ooo.ooo

10,000,000

Cell Number per Tube

Fig. 3. Inverse relationship ‘II-1 cells were centrifuged

behveen transduction efficiency and cell number per tube. Varying numbers of in 2 ml of a 1 :4 supe mixture in 15.ml culture tubes at 1500 g for 1 h in a Sorvall 6000B table-top centrifuge (lower curve), or in 1 ml of a 1: 1 supe mixture in cryotubes at 5000 g for 1 h (upper curve). In the latter experiment, comparison was made between centrifugation in the Eppendorf 5415C microcentrifuge and the Sorvall RCSC high-speed centrifuge. Linear regression of the log transformed values from both sets of samples was used to derive the indicated exponential equation. Samples were split after centrifugation to match the cell concentration of the sample with the lowest cell number and expanded for 5 days prior to harvest for enzyme assay.

This suggests that the relationship between cell number and transduction efficiency in both experiments may have a common geometric explanation (see Discussion). Centrifugation at 1500 g for increasing time periods up to 7 h resulted in a nearly linear increase in transduction (Fig. 4). With 3T3 cells, a linear increase had been observed for times up to 1 h (not shown). Higher centrifugal force was found to improve transduction up to approximately 10,000 g, after which no further gain was obtained (Fig. 5). Parallel curves were

300 1

01

, 0

1

2

3

Centrifugation

Fig. 4. Direct relationship

4

6

6

7

6

Time (hours)

between transduction efficiency and time. 3 X 10’ TF-1 cells were centnfuged for different time periods in 2 ml of a 1: 4 supe mixture at 1500 g in 15-ml culture tubes.

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-v-Noninfected

0

10000

20000

30000

40000

Relative Centrifugal Force (xg) Fig. 5. Direct relationship between transduction efficiency and relative centrifugal force plateaus at 10,000 g. 1 X lo5 TF-1 cells were aliquotted per cryotube in 1 ml of 1: 1 and 1:4 supe mixtures. The tubes were centrifuged in the Sorvall RCSC high-speed centrifuge for 1 h at the speeds indicated. Error bars indicate the range between duplicate samples.

obtained for two different strengths of supernatant, which argues against the plateau being caused by a saturation effect resulting, for example, from complete occupation of cell receptors by virus or from competition for transcription or expression factors. Measurement of cell numbers and viability showed that centrifugation for 2 h at 20,000 g had no significant effect on cell viability and growth (data not shown). These data indicate that centrifugation at 10,000 g is maximally effective and provides a margin of safety for TF-1 cells. 3.3. Transduction

of CD34 +

enriched

cells

Centrifugal enhancement has been applied to human CD34 + cells with very promising results. In two experiments, cord blood CD34 + cells were transduced over a 3-day period by three 2-h infections at 2400 g following 1 day of prestimulation in cytokine-containing medium (see Materials and methods). Between 1 and 3 weeks after infection, portions of the samples were harvested for enzyme assay. The enzyme activity of the centrifuged samples was about 6 times the normal levels of enzyme activity in non-transduced samples following centrifugation compared with levels up to two times the controls without centrifugation (Fig. 6). In previous studies we have observed up to 3 times control levels in cord blood CD34 + cells transduced without centrifugation (Bahnson et al., 1994), and the cause of the unusually low transduction in the non-centrifuged sample in the first experiment is unknown. In the second experiment, a portion of the sample was separated after the first and second infections, demonstrating the incremental effect of each individual infection.

4. Discussion Viruses centrifugal

are being engineered into vehicles for gene transfer, and the method of enhancement of transduction is a supplemental technology which may have

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T q

Noncentrifuged

0

Noninfected

0

Experiment

1

Experiment

2

Fig. 6. Centrifugal enhancement of transduction of cord blood CD34 + cells. Cord blood cells were transduced following 1 day of prestimulation in cytokine-containing medium (see Materials and methods). Bars represent the mean and S.E.M. for n = 7 (centrifuged) or n = 2 (conventional) samples harvested between 1 and 3 weeks after transduction. Cells were expanded in IL-3, IL-6, and SCF-containing medium. In Expt. 2, a portion of the sample was removed prior to the second and third transductions, and these cells were expanded and analyzed to show the incremental effect of each exposure.

broad application in the use of viral vectors for gene transfer purposes. Recently, Kotani and coworkers reported improved transduction of adherent and non-adherent cell targets with a variety of retroviral vectors using a 32°C centrifugation for 90 min and a subsequent 32°C incubation period prior to replacement of the medium (Kotani et al., 1994). We have more fully investigated several factors effecting centrifugal enhancement, and the results presented here should help to design optimized protocols for these applications. In particular, we have shown that effective transduction is: (1) achieved at room temperature; (2) directly related to time of centrifugation and to relative centrifugal force up to 10,000 g; (3) independent of volume of supernatant for volumes of 0.5 ml or greater using non-adherent cell targets in test tubes, but dependent upon volume for coverage of adherent cell targets in flat bottom plates; and (4) inversely related to cell numbers per tube using non-adherent cells. Maximum overall transduction might be obtained by maximizing the relevant factors. However, it is not yet known whether these may be independently varied to yield the highest possible transduction efficiencies. For example, under sufficiently high centrifugal forces, virus migration from the suspension to the walls of the tube may induce inactivation, which could alter the relationship between transduction and time of centrifugation from that observed under relatively low centrifugal forces. Additional experiments will be required to determine the extent to which centrifugal enhancement of transduction can be fully optimized, although the data suggest that further improvement over that observed in this study for CD34 + enriched cells may be obtained by increasing either the centrifugal force or the time of centrifugation. The inverse relationship observed for transduction efficiency and cell number may result from crowding or layering of cells in the cell pellet during centrifugation. If, as suggested by these data, exposure of the cell surface to the viral vector-containing

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medium is necessary for transduction, this implies that total transduction, as measured by the enzyme assay, is directly related to the exposed surface area of the cell pellet, which varies as the square, d2, of a dimension, d. The volume of the cell pellet is directly related to the cell number, which varies as the cube, d3. Specific enzyme activity, is expressed as units of fluorescent product, U, per milligram, of total protein. Since fluorescent product is directly related to total transduction, which is proportional to the exposed surface, d2, and since milligrams of protein is directly related to cell number, which is proportional to d3, it follows that enzyme activity, U/mg, is related to d2/d3 = l/d. Since cell number, N, is proportional to d3, d is proportional to N’13. Layering or crowding of cells reduces the surface to volume ratio, and the resulting decline in enzyme activity would be expected to be proportional to 1/N’13, i.e., enzyme activity is expected to decline in inverse proportion to the cube root of increasing cell numbers. The observed exponent for the regression line shown in Fig. 3 was -0.37, which is very close to the theoretical -0.33 discussed here as a first approximation. In any case, the relationship observed implies that multiple tubes or other means of increasing exposure of the cells to the virus-containing supernatant should be employed to maximize transduction for large numbers of cells. Theories for the mechanism of centrifugal enhancement of viral transduction may be divided between those suggesting an effect on the cells and those in which the centrifugal force acts primarily on the virus particles. Since the effect for some viruses can be observed even at fields below 100 g (Hodgkin et al., 19881, and since much higher forces are used to bring viruses out of suspension (e.g. 12,000 g for 5-16 h (Cepko, 1992)), it is reasonable to seek an explanation in terms of an effect on the cells. In an effort to uncover some ‘physiological’ or ‘biochemical’ role of centrifugation, Hudson (1988) systematically examined cell cycle, cell structure, and the influence of various factors known to affect cell metabolism, gene regulation, cell growth, morphology, etc. No consistent effect from any factor tested on centrifugal enhancement relative to non-centrifugal infection was found. Alternatively, an explanation proposed by Hodgkin et al. (1988) focuses on the reversibility of virus-to-cell binding. A two-phase process of infection was envisioned in which the first phase was reversible and the second phase was irreversible. These investigators proposed that centrifugation works on the first phase of reversible binding; (1) it increases the rate of virus association; (2) it decreases the rate of dissociation; and (3) by increasing the length of time each virus particle is bound, it increases the probability of virus being taken into the cell. Support for this mechanism included the demonstration of rapid dissociation of murine cytomegalovirus following replacement of the virus-containing medium with fresh medium, a much reduced effect of centrifugation when performed as early as 5 min after removal of virus-containing medium, and increased numbers of reversible plaques with increased time of centrifugation up to 15 min. Hodgkin et al. (1988) also demonstrated a dependence upon the direction of the force by inverting a flask in the centrifuge so that the cell monolayer faced away from the centrifugal field. This resulted in a 3-fold ‘disenhancement’, or reduction, of plaque formation in relation to exposure at 1 g for the same period. Direct dependence upon centrifugal force for MCMV plaque formation at speeds up to 1000 g was also

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demonstrated. In our study, using a retroviral vector and TF-1 cells, we have extended the upward boundary of dependence upon centrifugal force up to 10,000 g, at which a plateau for transduction was observed (Fig. 5). Taken together, these data demonstrate a continuous dependence upon the direction and magnitude of the gravitational field from negative effect with the field oriented away from the cells to a maximum highly positive effect for fields oriented toward the cells. Virus particles in suspension are not significantly affected by low gravitational fields in relation to random forces of diffusion. Even under centrifugal fields of 1000 g, virus remains suspended. However, a virus particle attached to a cell receptor has a reduced energy and remains associated with the receptor until the magnitude and direction of the kinetic forces are sufficient to overcome the energy of association. (Less frequently, a virus passes from the reversible phase of attachment to an irreversible second phase in the process of infection.) When the applied centrifugal field is oriented toward the cell surface, the centrifugal field contributes a component of force which is opposite in direction to the resultant force leading to dissociation. It follows that virus remain attached for longer periods of time, which, as proposed by Hodgkin et al. (19881, increases the probability of entry of the virus into the cell. Conversely, orientation of the field in the opposite direction results in shorter average periods of attachment and decreased probability of entry of virus into the cell. In this model, centrifugation acts through its effect on the virus particle in the direction of the cell, and the magnitude of this effect should be proportional to the magnitude of the centrifugal force upon the virus relative to the resultant force required for dissociation. This may imply that at the plateau observed at 10,000 g for a retroviral vector, the forces leading to dissociation are completely counteracted by the centrifugal field. Since virus may be pelleted by centrifugation at 12,000 g (Cepko, 19921, the forces acting at 10,000 g are feasibly sufficient to overcome the forces of diffusion which maintain the virus in suspension. This implies that the forces of association are relatively weak in relation to the forces of diffusion, and that under normal gravity, with polybrene present, only a relatively small proportion of the viruses are attached to receptors. In other words, the equilibrium is much favored toward the dissociated state. This is also supported by the observation that centrifugation can yield lo-fold or greater increases in transduction efficiency in comparison to exposure for the same time period at normal gravitational fields. The independence of transduction efficiency upon volume, found also by Hodgkin et al. (1988) under non-centrifugal conditions, brings into question the concept of ‘multiplicity of infection’ (m.o.i.1. For specifying infection conditions, the term IM may be preferable to the more commonly used term, m.o.i., as a measure of the presumed input ratio of virus particles to target cells in an infection protocol. IM is directly dependent upon the titer and added volume of virus-containing medium, whereas m.o.i. is more properly an observed efficiency of infection ‘after the fact’ and is thus equivalent to transduction efficiency. As shown here (Fig. 11, transduction efficiency is dependent upon virus concentration rather than upon volume (or number of viruses) added per target cell. Consequently, increasing the IM by using greater volumes does not effectively increase the transduction efficiency for this vector. Furthermore, since transduction efficiency is not linear with virus concentration (Fig. 11, the calculated titer (infectious particles per ml) is not a constant, but increases with increasing dilution.

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These problems complicate use of the convenient terms, ‘titer’ and ‘m.0.i.‘; although this situation may be different with virus types which bind irreversibly. The results described here confirm and extend the promise for centrifugal enhancement of retroviral mediated gene transfer reported by Kotani et al. (1994). This method is being further investigated in our laboratory for application to gene transfer into CD34 + enriched hematopoietic cells as part of our preclinical study for gene therapy for Gaucher disease. Numerous other applications may be forthcoming. By substantially reducing the time required for efficient transduction, centrifugation may open possibilities for ‘pulsing’ target cells in order to study time-dependent processes involved in infection. It may provide better sensitivity for assay and detection of low level replication competent retroviruses in clinical grade gene therapy products. Centrifugal enhancement is likely to improve transduction using other types of viral vectors, based upon previous studies with wild-type viruses (Hodgkin et al., 1988). We would expect the method to enhance infection for virus types in which a reversible phase of attachment is characterized by an equilibrium which strongly favors the dissociated state, allowing counteraction of dissociation by the centrifugal field.

Acknowledgements The authors wish to thank Amy Kemp and Jason Lancia for technical assistance and Marge Jasko for help with the manuscript. The generosity of Immunex Corporation for the gifts of cytokines (IL-3, IL-6, SCF, and GM-CSF) of Paul Robbins and Toya Onashi for the MFG-GC vector, and of CellPro, Inc. for immunoaffinity columns is also appreciated. This work was supported by Grants DK 43709 and DK 44935.

References Bahnson, A., Nimgaonkar, M., Fei, Y., Boggs, S., Robbins, P., Ohashi, T., Dunigan, J., Li, J., Ball, E.D. and Barranger, J.A. (1994) Transduction of CD34 + enriched cord blood and Gaucher bone marrow cells by a retroviral vector carrying the glucocerebrosidase gene. Gene Ther. 1, 176-184. Cepko, C. (1992) Large scale preparation and concentration of retrovirus stocks. In: F.M. Ausabel et al. (Ed%), Current Protocols in Molecular Biology, Suppl. 17. Greene Publishing Associates, and John Wiley & Sons, Sect. 9.12.1-9.12.5. Danos, 0. and Mulligan, R.C. (1988) Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc. Natl. Acad. Sci. USA 85, 6460-6464. Deisseroth, A.B., Zu, A., Claxton, D., Hanania, E.G., Fu, S., Ellerson, D., Goldberg, L., Thomas, M., Janicek, K., Anderson, W.F., Hester, J., Korbling, M., Durett, A., Moen, R., Berenson, R., Heimfeld, S., Hamer, J., Calve& L., Tibbits, P., Talpaz, M., Kentarjian, H., Champlin, R. and Reading, C. (1994) Genetic marking shows that PH+ cells present in autologous transplants of chronic myelogenous leukemia contribute to relapse after autologous bone marrow in CML. Blood 83, 3068-3076. Dunbar, C.E., O’Shaughnessy, J.A., Cottler-Fox, M., Carter, C.S., Doren, S., Cowan, K.H., Leitman, SF., Wilson, W., Young, N.S. and Nienhuis, A.W. (1993) Transplantation of retrovirally-marked CD34 + bone marrow and peripheral blood cells in patients with multiple myeloma or breast cancer. Blood 82 (Suppl. 1). 217a. Ho, W.-Z., Cherukuri, R., Ge, H.-D., Curilli, J.R., Song, L., Whitko, S. and Douglas, S.D. (1993) Centrifugal enhancement of human immunodeficiency virus type 1 infection and human cytomegalovirus gene expression in human primary monocyte/macrophages in vitro. J. Leukocyte Biol. 53, 208-212.

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