La chromatographie est considérée comme étant la technologie la plus prometteuse pour la purification à grande échelle des vecteurs viraux. Les auteurs ont précédemment démontré que la chromatographie d’affinité sur colonne héparine pouvait être utilisée avec succès pour purifier les vecteurs dérivés du virus Moloney de la leucémie murine (MoMLV) pseudo-typés avec VSV-G, donnant d’excellents résultats en terme de récupération de particules actives, reproductibilité et sélectivité. Dans cette étude, les auteurs ont examiné si la capacité des particules retrovirales de se lier spécifiquement à l’héparine était restreinte au pseudo-type VSV-G produit dans les cellules 293. Il a été démontré que les particules rétrovirales déficientes en VSV-G étaient capturées par chromatographie d’affinité sur colonne héparine aussi efficacement que les particules contenant VSV-G. Également, les particules rétrovirales pseudo-typées avec RD114, dérivées de la lignée cellulaire HT1080, se sont liées à l’héparine avec la même affinité que les pseudo-types VSV-G dérivés des cellules 293, avec des récupérations de particules fonctionnelles de 43%. Ces résultats indiquent que la chromatographie d’affinité sur colonne d'héparine peut être utilisée pour purifier des vecteurs rétroviraux produits par différentes lignées cellulaires indépendamment de la protéine rétrovirale d’enveloppe utilisée pour le pseudo-typage. Les implications de ces découvertes sur le mécanisme d’attachement des vecteurs sur les cellules cibles et le design des vecteurs rétroviraux reciblés pour la thérapie génique sont discutés.

Chromatography is deemed the most promising technology for large-scale purification of viral vectors. The authors have previously shown that heparin affinity chromatography could be successfully employed for the purification of VSV-G pseudotyped Moloney murine leukemia virus (MoMLV)-derived vectors giving excellent results in terms of recovery of active particles, reproducibility and selectivity. In this study, the authors examined whether the ability of retrovirus particles to specifically bind to heparin ligands is restricted to VSV-G pseudotypes produced by 293-based packaging cells. It is shown that VSV-G deficient retrovirus particles are captured by a heparin chromatography column as efficiently as VSV-G containing particles. Most strikingly, RD114 pseudotyped retrovirus particles derived from a HT1080-based cell line were found to bind heparin with the same affinity as 293-derived VSV-G pseudotypes. RD114 pseudotyped retrovirus particles were successfully isolated using heparin affinity chromatography obtaining good recoveries of functional particles (43%). These results indicate that heparin affinity chromatography can be extended to the purification of retroviral vectors produced by different packaging cell lines independently of the Env-protein used for pseudotyping. The implications of these findings on mechanism of vector attachment to target cells and the design of retargeted retroviral vectors for gene therapy are discussed.

Retroviral vectors constitute a valuable tool for gene transfer technology. The wide clinical application of these vectors for gene therapy will depend on the availability of efficient large-scale manufacturing procedures useful for the purification of alternative vector pseudotypes. Retroviral vectors are labile enveloped viruses that require the strategic design of gentle purification processes. Chromatography plays an important role in the purification of high value bioproducts since it enables fast, efficient, reproducible and selective separations. Not surprisingly, chromatography is becoming the method of choice for the large-scale purification of most gene therapy vectors including retroviral vectors (Debelak et al., 2000; Kaludov et al., 2002; Zolotukhin et al., 2002; Arcand et al., 2003; Smith et al., 2003; Davidoff et al., 2004; Segura et al., 2005).

Chromatography separates retroviruses from impurities contained in the vector supernatant by exploiting physical and biochemical features of retrovirus particles. For instance, using size exclusion chromatography scientists can take advantage of the large size of retroviruses (~100 nm) for its separation from contaminating proteins and other low molecular weight impurities (McGrath et al., 1978; Slepushkin et al., 2003; Transfiguracion et al., 2003). Additionally, the negatively charged surface of retroviruses can also be exploited for purification purposes by utilizing positively charged functional groups, such as those found in anion exchange (or hydroxyapatite) resins, that strongly bind retrovirus particles (Smith and Lee, 1978; Prior et al., 1995; Kuiper et al., 2002; Scherr et al., 2002; Yamada et al., 2003). On the other hand, the specific recognition of molecular structures on the viral membrane by affinity ligands would allow the selective isolation of retrovirus particles using affinity chromatography. Due to its high resolution, affinity chromatography offers the potential to reduce the number of purification steps increasing product yields and decreasing process costs. However, to take full advantage of this technology it is important to identify stable, inexpensive and versatile affinity ligands that specifically bind retrovirus particles. Unfortunately, little is known about the composition of the retroviral membrane, which complicates the selection of appropriate affinity ligands. Retroviral vectors are frequently genetically modified to contain Env-proteins of other viruses giving rise to a variety of vector pseudotypes. In addition, MoMLV particles are known to randomly incorporate various host-derived proteins on their membrane (Hammarstedt et al., 2000) that remain largely unidentified. Therefore, the composition of the viral membrane is expected to vary to some extent depending on the cell line used for vector production. As a consequence, it seems difficult to find an affinity ligand useful for the purification of all retroviral vectors, which would be highly desirable to simplify and unify vector manufacturing procedures.

In principle, retroviral vectors could be purified by immunoaffinity chromatography by relying on the specific interaction between immobilized antibodies and the viral Env-protein. However, the high costs associated with antibody purification and immobilization, the low stability of these ligands towards sanitizing agents and the harsh conditions usually required to break antibody-antigen interactions do not favor the use of this method for large-scale purification of retroviral vectors (Andreadis et al., 1999). Moreover, depending on the Env-protein used to pseudotype the vector, chromatography columns and protocols should be specifically designed for each individual case. Another possibility is to engineer vectors to contain affinity tags inserted on the surface of the virus to facilitate their purification. Hexahistidine affinity tags have been inserted into the MoMLV ecotropic Env-protein to allow purification by immobilized metal affinity chromatography (IMAC) (Ye et al., 2004). Additionally, chemically biotinylated retrovirus particles have shown to bind streptavidin coated adsorbents in batch experiments (Williams et al., 2005). However, engineering vectors by inserting tags or chemically modifying the Env-protein without reducing or eliminating the viruses’ ability to transduce cells has proved to be a difficult task as demonstrated by many unsuccessful efforts to alter the structure of Env-proteins for targeting purposes (Katane et al., 2002; Palù et al., 2000; Tai et al., 2003).

An attractive alternative is to explore the natural ability of these viruses to bind commercially available affinity ligands or immobilized viral receptors. Heparin is a relatively inexpensive and stable affinity chromatography ligand used to purify a variety of biomolecules and viruses. Heparin structurally mimics the widely distributed heparan sulfate cell surface proteoglycan which has been recognized as a receptor for attachment of numerous viruses including herpes simplex virus (both HSV-1 and HSV-2) (WuDunn and Spear, 1989; Shieh et al., 1992; Spear et al., 1992), foot and mouth disease virus (FMDV type O) (Jackson et al., 1996; Chen et al., 1997), dengue 2 virus (Chen et al., 1997) and adeno-associated virus (AAV-2) (Summerford and Samulski, 1998). For these viruses, heparin affinity chromatography constitutes a valuable tool for purification and serves to study virus-heparin interactions (Navarro del Cañizo et al., 1996; O'Keeffe et al., 1999; Zolotukhin et al., 1999). It is interesting to note that for most viruses, including the ones mentioned above, the heparin-binding domains on the virus responsible for virus-heparin interaction were found to be localized on viral-encoded proteins (Herold et al., 1991; Chen et al., 1997; Fry et al., 1999; Kern et al., 2003; Opie et al., 2003). Heparan sulfate proteoglycan has also been implicated as a receptor for some retroviruses, namely human immunodeficiency virus (HIV-1) and Friend murine leukemia virus (F-MuLV) for which heparin-binding sites responsible for the virus-heparin interaction were also identified within specific domains of the wild-type Env-protein (Roderiquez et al., 1995; Mondor et al., 1998; Cladera et al., 2001; Jinno-Oue et al., 2001; Vives et al., 2005).

Previous studies have shown that heparin affinity chromatography was a useful method for the purification of VSV-G pseudotyped retroviral vectors derived from 293 producer cells giving excellent results in terms of yield, selectivity and reproducibility (Segura et al., 2005). Elution of retrovirus particles from heparin affinity columns was achieved under mild conditions (neutral pH and 0.35 M NaCl) resulting in high recoveries of infective particles (61%). However, the extended applicability of heparin affinity chromatography to the purification of different retroviral vector pseudotypes or vectors produced by different cell lines remained unclear. To further characterize retrovirus-heparin interactions, the authors examined the ability of VSV-G deficient retrovirus particles as well as RD114 pseudotyped particles produced by a different packaging cell line, the FLYRD18 which is a HT1080-based cell line, to bind immobilized heparin ligands.

Packaging cell lines and retroviral vectors

Two packaging cell lines that produce Moloney murine leukemia virus (MoMLV) vector particles psedudotyped with the envelope glycoproteins of either vesicular stomatitis virus VSV-G (293GPG) or cat endogenous virus RD114 (FLYRD18) were used. The 293GPG packaging cell line, derived from 293 human embryonic kidney cells (Ory et al., 1996), was stably transfected to generate a retroviral vector encoding a fusion protein that links the simplex virus thymidine kinase protein (TK) with the green fluorescent protein (GFP) (Paquin et al., 2001). This cell line, a generous gift from Dr. J. Galipeau (Lady Davis Institute for Medical Research, Montreal, QC, Canada), was adapted to grow in suspension culture (Ghani et al., submitted for publication). The stable FLYRD18 packaging cell line derived from HT1080 human fibrosarcoma cells (Cosset et al., 1995) produces GFP3 vector (Qiao et al., 2002). These cells and the 143B target cells were graciously provided by Dr. M. Caruso (Centre de recherche en cancérologie de l’Hôtel-Dieu, Université Laval, Québec, QC, Canada). Cells were maintained in tissue culture flasks in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO-BRL, Gaithersburg, MD) supplemented with 10% heat-inactivated foetal bovine serum (FBS; HyClone, Logan, UT) at 37°C, 100% humidity and a 5% CO₂ atmosphere. 293 GPG culture medium additionally contained tetracycline (1 µg/mL; Fisher Scientific, Nepean, ON, Canada) to repress the expression of VSV-G gene.

Retroviral vector production

VSV-G pseudotyped vector production was carried out in a 250 mL shake flask (50 mL working volume) inoculated at 2×10⁵ 293-GPG cells/mL. Cells were grown in calcium free DMEM supplemented with 10% FBS and tetracycline until the cell density reached 2×10⁶ cells/mL. At this point VSV-G expression was induced by entirely removing the tetracycline-containing medium by centrifugation of the cell culture (420×g, 10 min). Cells were washed with phosphate-buffered saline pH 7.4 (PBS) and the cell pellet was resuspended in fresh tetracycline-free medium, re-introduced into the shake flask and incubated at 37°C during 48 h. In parallel, the production of Env-protein deficient retrovirus particles was carried out following the same protocol with the exception that the cells were resuspended in fresh tetracycline-containing medium. Retrovirus containing supernatants were harvested every 24 h during 5 days by centrifugation of the cell culture (420×g, 10 min) and replaced with fresh medium. RD114 pseudotyped vector particles were produced in 175 cm² tissue culture flasks (35 mL working volume) by FLYRD18 adherent cells. Cells were seeded at a density of 4×10⁵ cells/mL and grown for 48 h in High Glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. The vector production phase was initiated at ~ 80% confluence (~8×10⁵ cells/mL) by washing the cells with PBS and replacing the medium with 35 mL of fresh medium. Retrovirus containing supernatant was harvested every 24 h during 5 days and replaced with fresh medium. Harvested retrovirus supernatants were clarified using 0.45 µm pore size syringe-mounted filters (Millipore, Bedford, MA) and concentrated 20-fold using a 76 mm diameter Omega^TM polyethersulfone membrane disc filter with a molecular weight cut-off of 300,000 (Pall Gelman Sciences) in a 400 mL stirred cell ultrafiltration unit (Amicon 8400; Millipore, Etobicoke, ON, Canada) as previously described (Segura et al., 2005). Retrovirus-enriched retentate was diafiltered against cold heparin affinity adsorption buffer (150 mM NaCl in 20 mM Tris-HCl buffer, pH 7.5). Virus stocks were aliquoted and stored at -80ºC.

Infective retroviral vector titer determination

Quantification of infective particles by flow cytometric analysis has been previously reported (Segura et al., 2005). Briefly, 293 (for HT1080-derived vector particles) or 143B (for 293GPG-derived vector particles) target cells were seeded in 6-well plates and exposed to 1 mL aliquots of serial dilutions of virus in DMEM containing 8 µg/mL of polybrene during 3 h at 37ºC. After addition of DMEM containing 20% FBS (1 mL), the cells were incubated for 48 h at 37°C under 5% CO₂ atmosphere. Transduced cells were washed with PBS, detached with trypsin-EDTA, fixed with 2% formaldehyde and resuspended in 1 mL of PBS. Samples were then subjected to fluorescent-activated cell sorting (FACS) analysis and viral titers were determined as previously described (Segura et al., 2005).

Quantitation of retrovirus particles by immunofluorescence microscopy

VSV-G pseudotyped and VSV-G deficient retrovirus particles were quantitated by immunofluorescence microscopy using a method adapted from Pizzato and collaborators (Pizzato et al., 1999). Virus samples were mixed with 100 nm red fluorescent carboxylate-modified microspheres (Molecular probes, Eugene, OR) at a final concentration of 5.4×108 spheres/mL. Mixtures (5 μL) were spread on a 1 cm² area of glass slide and air dried at room temperature for 30 min. Virus particles were fixed with 2% formaldehyde during 15 min, washed 5 times with PBS and permeabilized with 0.2% Triton X-100 for 15 min at room temperature. Slides were washed once with PBS, blocked for 15 min with 10% FBS in PBS and washed 3 times with PBS. Gag and VSV-G immunofluorescence staining was performed for each sample separately. Samples were incubated for 45 min at room temperature with primary antibodies, either rat polyclonal anti-Gag in house antibody or a monoclonal antibody against the envelope protein (mouse Mab anti-VSV-G; Roche Diagnostics, Indianapolis, IN). Slides were washed 3 times with PBS and incubated with appropriate secondary antibodies conjugated with fluorophores, either Alexa Fluor 488 goat anti-rat antibody (Molecular probes) or FITC F(ab’)2 goat anti-mouse antibody (Serotec, Oxford, UK). After 45 min of incubation with secondary antibodies at room temperature, the slides were washed once with PBS, air dried and mounted with slow-fade mounting solution (Molecular Probes). Pictures were taken using a Princeton Instruments CCD camera mounted on a Leitz Aristoplan upright fluorescence microscope. The Gag+ or VSV-G + particle concentration were estimated based on the ratio between immunostained virus particles and fluorescent microspheres.

Effect of soluble heparin on VSV-G retrovirus transduction to 143B target cells

Equal volumes of retrovirus supernatant were incubated for 30 minutes at 37°C in the presence of various concentrations of heparin (Sigma). A negative control was incubated without heparin. Following virus treatment with heparin, titers were determined for triplicate experiments at each concentration of heparin.

Heparin affinity chromatography

Chromatography was performed using a low-pressure liquid chromatography system (GradiFrac; GE Healthcare, Uppsala, Sweden) at room temperature and monitoring protein elution by UV absorbance at 280 nm. All samples were filtered with a 0.45 µm GHP Acrodisc filter membrane (Pall Gelman Sciences) prior to chromatography. The ability of 293-derived VSV-G deficient retrovirus particles to bind immobilized heparin ligands was investigated using a previously defined heparin affinity chromatography step-wise elution strategy (Segura et al., 2005). Briefly, a 1-mL Fractogel^® EMD Heparin (S) column was pre-equilibrated with 150 mM NaCl in Tris-HCl buffer, pH 7.5 and loaded with 3 mL of sample. A step-wise NaCl elution strategy consisting in a wash step at 150 mM NaCl (19.5 column volumes [CV]), an elution step at 350 mM NaCl (13 CV) and a high stringency final wash step at 1200 mM NaCl (7.5 CV) was applied. The running linear flow rate was 153 cm/h. Fractions from each peak were pooled and analyzed by immunofluorescence microscopy. The binding aptitude and affinity of 293-derived VSV-G pseudotyped and HT1080-derived RD114 pseudotyped retrovirus particles was compared using a linear NaCl gradient elution strategy. Briefly, the same protocol described above was followed with the exception that following the wash step at 150 mM NaCl, a linear gradient was applied from 150 to 1150 mM NaCl in Tris-HCl buffer, pH 7.5, at a rate of 50 mM NaCl/min and a linear flow rate of 92 cm/h. Fractions of 2.5 mL were collected throughout the run and immediately tittered. In addition, the recovery of HT1080-derived RD114 pseudotyped retrovirus infective particles using the step-wise strategy was estimated by flow cytometry analysis of the pool of fractions from each peak.

The effect of heparin on VSV-G retrovirus transduction to target cells

Retrovirus supernatants were incubated in the presence of various concentrations of heparin as described in Materials and Methods. A negative control was incubated without heparin. Infectious titers were determined by flow cytometric analysis for GFP expression. Titer values presented are the mean ± standard deviation of triplicate samples. Abbreviations: IVP infective virus particles

Although these experiments suggest a possible interaction between the virus and the heparin molecule, they are not conclusive. Polybrene and other polycations are generally believed to exert their enhancing effects on retrovirus transduction by reducing the electrostatic repulsion between retroviruses and cells; thus, increasing retrovirus binding to target cells. Since heparin is a highly sulfated linear polysaccharide, it could be mistakenly inferred that the inhibitory effect heparin has on vector transduction results from electrostatic interference as a consequence of its polyanionic nature that would repel both, the negatively charged viruses and cells. However, heparin affinity ligands attached to a chromatography matrix have shown to efficiently capture retrovirus particles while the same matrix carrying anionic sulfate groups failed to do so which clearly indicated that a specific interaction between the virus and heparin is taking place, rather than repulsion (Segura et al., 2005).

Interestingly, treatment of MoMLV particles with low concentrations of heparin (1 U/mL) enhanced transduction by 21% compared to non-treated virus particles. A similar concentration dependent dual effect (enhancement and inhibition) of soluble heparin on the infectivity of F-MuLV has previously been reported (Jinno-Oue et al., 2001). The authors explained this effect of heparin by presenting a model in which the heparin molecule serves as a molecular bridge between the heparin-binding domains identified on this virus and heparin-binding structures at the cell surface. According to this model, low concentrations of soluble heparin are expected to enhance virus infectivity by acting as a bridge between the virus and the cells whereas high concentrations of heparin would inhibit virus infection by blocking binding sites on the virus and the cells.

VSV-G deficient retrovirus particles interact with heparin ligands

Supernatants were produced in parallel with or without the addition of tetracycline generating comparable concentrations of VSV-G deficient retrovirus particles and VSV-G containing particles respectively. Concentrated virus stocks were loaded onto a Fractogel^® EMD Heparin (S) column and elution was carried out using a step NaCl gradient as described in Materials and Methods. Heparin purified fractions eluting at 350 mM NaCl were collected, pooled and Gag+ particles were quantified by immunofluorescence microscopy. Values presented are the mean ± standard deviation of 5 counts.

293 and HT1080-derived retrovectors bind heparin with the same affinity

VSV-G and RD114 pseudotyped concentrated virus stocks were loaded onto a Fractogel^® EMD Heparin (S) column and elution was carried out using a linear NaCl gradient (150-1150 mM) as described in Materials and Methods. Eluting fractions were collected throughout the run and subjected to flow cytometry analysis for GFP expression. Values presented are the mean ± standard deviation of duplicate samples.

3 mL of a 20-fold concentrated virus containing 2.2×10⁶ IVP/mL were loaded onto a 1 mL Fractogel^® EMD Heparin (S) column. The virus was eluted by addition of 350 mM NaCl into the mobile phase. A similar chromatographic behavior was previously observed for VSV-G pseudotyped particles. Retrovirus particles were recovered in a defined peak (4.5 mL) containing 6.2×10⁵ IVP/mL. Abbreviations: IVP infective virus particles.

Recovery of RD114 pseudotyped retroviral vector in fractions eluted from the Fractogel^ EMD Heparin (S) column using the developed step-wise elution strategy. Titer values presented for each fraction are the mean of duplicate determinations. Average titer and recovery values for the 3 runs are shown in the table. Abbreviations: IVP infective virus particles; sd standard deviation.

Previous studies from our laboratory have shown that VSV-G pseudotyped MoMLV-derived vectors stably interact with heparin. In this work we demonstrate that both VSV-G deficient and RD114 pseudotyped retrovirus particles can also bind heparin with similar efficiency and affinity as VSV-G pseudotyped particles. Therefore, these results indicate that contrary to what was reported for most viruses that interact with heparin, the viral Env-protein (VSV-G in our case) does not appear to be involved in retrovirus-heparin interactions. Instead, most likely unidentified cellular component(s) on the virus surface play a role for the observed heparin-binding activity. Thus, in principle the method can be extended to the purification of 293 and HT1080-derived retrovectors regardless of the Env-protein carried by the virus.

Moreover, the fact that both 293 and HT1080-derived retrovectors bind heparin with identical affinity suggests that the heparin-binding activity probably derives from a common component ubiquitously distributed in different packaging cell types. In this case, the host-derived component would be incorporated into various vectors opening the possibility of extending the use of heparin affinity chromatography to the purification of potentially all MoMLV-derived vectors regardless of the cells from which they were derived or Env-protein used for pseudotyping. The usefulness of heparin affinity chromatography for the purification of AAV vectors has been severely compromised by the fact that unlike AAV-2 which stably interacts with heparin, other AAV serotypes (i.e. AAV-1, AAV-4 or AAV-5) lack heparin-binding activity (Kaludov et al., 2002; Zolotukhin et al., 2002; Zolotukhin, 2005). In view of this limitation with AAV vectors, the possibility of extending the method to the purification of all MoMLV-derived vectors is very attractive.

Retrovirus-heparin interaction is stable but reversible requiring relatively low salt concentrations for dissociation. This is important considering the susceptibility of retroviruses to osmotic pressure (Aboud et al., 1982; Andreadis et al., 1999; Segura et al., 2005). The recovery of infective particles was higher for VSV-G pseudotyped vectors (61.1%) than for RD114 pseudotyped vectors (42.6%). This result could reflect the poorer stability of RD114 Env-protein or could result from suboptimal vector titers in the starting material. Nevertheless, we would like to point out that to date heparin affinity chromatography has given the highest recoveries of infective particles for MoMLV-derived vectors; possibly because other adsorptive chromatography methods require harsher conditions for the elution of virus particles from the chromatography columns including the addition of noxious desorption reagents (d-biotin and imidazole) and the use of higher ionic strengths, all of which were shown to affect the vectors’ stability (Kuiper et al., 2002; Ye et al., 2004; Segura et al., 2005; Williams et al., 2005).

The Env-protein-receptor interaction is considered the main mechanism of MoMLV vector attachment and entry to target cells. More recently, several studies suggested that at early steps of retrovirus transduction, initial virus adsorption via unidentified co-receptors at the cell surface may occur independently of the Env-protein and viral receptors. The incorporation of host-derived heparin-binding components and/or cell adhesion molecules on the virus surface would allow virus particles to use cell-cell and/or cell-matrix interaction mechanisms for attachment to target cells prior to membrane fusion and entry. Abbreviations: HSPG heparan sulfate proteoglycan; HS heparin sulfate chains.

The authors wish to thank Dr. Manuel Caruso, Normand Arcand and Gavin Whissell for careful review of this manuscript and helpful discussions. We appreciated the help of Pierre Trudel with fluorescence microscopy, Andre Migneault with image files and Lucie Bourget with FACS analysis. This work was supported by a NSERC Strategic Project grant and the NCE Canadian Stem Cell Network.

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