Title: Method to Improve Viral Infection FIELD OF THE INVENTION
The invention relates to a method to improve the viral infection of target cells. The method can be used to enhance gene therapy protocols by enhancing retroviral transduction of cells. BACKGROUND OF THE INVENTION
Retroviral vectors are well suited vehicles for gene transfer because they can stably integrate into the chromosomes of infected target cells at high efficiency. In addition, they do not direct the synthesis of viral proteins thus circumventing the problem of the transduced cells being removed by virus-specific immune responses of the host. The main parameters that are thought to limit retroviral transduction efficiencies are: the low susceptibility of the target cells to retroviral infection, including the level of expression of retrovirus receptors (Orlic, D., et al., 1996; Kavanaugh, M. P. et al., 1994); the proliferative activity of the target cell immediately post-infection (as integration of viral DNA depends on mitosis) (Agrawal, Y. P. et al., 1996; Ponchio, L. et al., 1995; Roe, T. Y. et al., 1993; Knaan-Shanzer, S. et al., 1996); and the ratio of infective virus particles relative to the target cells (multiplicity of infection: MOI). However, even under conditions with a relatively high MOI, transduction efficiencies can remain low, partly because of equipolar anionic surface charges of retrovirus envelope proteins and mammalian cell membranes (Toyoshima, K. and Vogt, P. K. 1969; Darnell, J. et al., 1986). This obstacle can be partially addressed by the addition of polycations to virus-containing cell-free medium (Ryser, H. J. 1967; Due-Nguyen, H. 1968). More recently, encapsulation of retroviruses in cationic liposomes was shown to allow infection of previously resistant cell lines and to enhance the retroviral transduction of murine fibroblasts and human fibrosarcoma cells (Faller, D. V. and Baltimore, D. 1984; Hodgson, C. P. and Solaiman, F. 1996). Another approach to facilitate the encounter of virus particles and target cells has exploited the ability of human plasma fibronectin (FN) to bind to virus (Hanenberg et al., 1996) as well as the β-1
integrins α4β: (VLA-4, CD49d/CD29) and a5pj (VLA-5, CD49e,CD29) which are expressed on many cells including primitive human hematopoietic cells (Kerst, J. M. et al., 1993; Teixido, J. et al., 1992; Hanenberg, H. et al., 1997). This has allowed the use of virus-containing supernatant infection protocols to replace co-cultivation to achieve equivalent hematopoietic stem cell transduction efficiencies (Conneally, E. et al., 1998; Dao, M. A. et al., 1998; van Hennik, P. B. et al., 1998; Schilz, A. J. et al., 1998).
There is a need to develop less cumbersome methods to improve retroviral transduction. SUMMARY OF THE INVENTION
The present inventors have developed a simpler strategy for improving retroviral transduction by increasing the probability of virus-target cell encounters using tissue culture dishes with a net positive charge or coated with a cation (such as poly-L-lysine) to increase virus presentation to the target cell by concentrating both the virus and the target cell on a common surface. The results show that this principle can be successfully applied to a variety of mammalian cells including human hematopoietic stem cells where it replaces the need for simultaneous exposure of the virus and cells to fibronectin. Accordingly, the present invention provides a method of improving viral infection of cells comprising:
(a) providing a vessel comprising a material with a net positive charge; and
(b) adding the virus and the cells to be infected to the vessel under conditions to allow the virus to infect the cells.
In a preferred embodiment, the virus is a retrovirus and the vessel is coated with a cation.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are
given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be described in relation to the drawings in which:
Figures 1A-D are graphs showing the time course of retrovirus adhesion to petri dishes coated with FN (A); poly-L-lysine (Lys), (B); glutamine (Glu) (C); or nothing (water = H20) (D). Figures 2A and B are bar graphs showing the adherence of xenotropic (Xeno) (A) and amphotropic (Ampho) (B) retrovirus particles on petri dishes as compared to tissue culture dishes coated with poly-L- Lysine, glutamine or fibronectin.
Figures 3A-F are FACS analyses of multi-lineage engraftment with green fluorescent protein (GFP) expression of human cord blood cells harvested from NOD/SCID mice. CD34-CD19+/20+ human lymphoid cells (panel A), were also analyzed for GFP expression (panel B), as were CD15+ mature human granulocytes (panel C) and total human hematopoietic (CD45/71+) cells (panel D). Panel E shows the engrafted cells from a mock transduction and panel F shows the level of antibody staining seen in a control (normal) NOD/SCID mouse (containing no human cells).
Figures 4A-C are graphs showing engraftment and transduction efficiency of lπv human CB cells injected into irradiated NOD/SCID mice: (A) engraftment of human CB cells after transduction with viral supernatant of PG13 cells on fibronectin coated petri dishes or uncoated tissue culture dishes; (B) the transduction efficiency into NOD/SCID repopulating cells was determined by dividing the number of GFP+/CD45+ by the number of CD45+ cells; (C) the calculated yield of transduced (GFP+) human (CD45+) cells per mouse for each transduction condition.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method of improving viral infection of cells comprising:
(a) providing a vessel comprising a material with a net positive charge; and
(b) adding the virus and the cells to be infected to the vessel under conditions to allow the virus to infect the cells.
The virus can be any virus and is preferably a recombinant viral vector. Accordingly, the invention can be used to improve viral transduction of cells, for example, to increase gene transfer.
The recombinant viral vector may be constructed using a variety of viruses which have been adapted as vectors, including adenoviruses, retroviruses, lentiviruses, pseudotyped viruses, DNA viruses and herpes viruses such as Herpes simplex virus. Preferably, the recombinant viral vector is constructed using a retrovirus. Retroviruses may be selected for a wide range of host target cells, including avian, mammalian and other animal cells. Moloney murine leukemia virus, myeloproliferative sarcoma virus and human immunodeficiency virus are some examples of suitable retroviruses. Viral vectors may be constructed from cloned retroviral cDNA using conventional techniques. A viral vector may be constructed by deleting certain genes required for viral replication from the viral genome, such as the gag gene which encodes for group specific antigens, the pol gene which encodes for reverse transcriptase and integrase and the env gene which encodes the envelope protein.
The viral vector typically (but does not necessarily) includes one or more exogeneous genes that may be ligated into the deleted genome of the virus. The exogenous gene(s) may include any gene (or DNA sequence) that one wants to introduce into the target cell. Often, the exogenous gene encodes a biologically active protein. A biologically active protein may be selected to modify the genotype and phenotype of the cell. For example, the exogenous gene may be selected for gene augmentation
to modify the expression of mutant genes in the cell, or to restore or alter gene function by introducing the exogenous gene into specific or non-specific sites in the cell's genome. The exogenous gene may be operatively linked to one or more expression control sequences. Sequences introduced into vectors can also be used as permanent cell tracers, or to control the expression of genes adjacent to the integrated vector, or to facilitate the later manipulation of transduced cell genomes.
In order to detect cell transduction, the retroviral vector may contain a marker. Examples of markers include proteins that confer resistance to toxic or inhibitory compounds such as neomycin, hygromycin, chloramphenicol, methotrexate, mycophenolic acid or various chemotherapeutic agents. In addition, detectable markers such as green fluorescent protein (GFP) may also be used.
The target cells can be any cell that one wishes to infect or transduce with a virus including prokaryotic and eukaryotic host cells, such as bacterial, mammalian, yeast or other fungi, viral, plant, or insect cells. In one embodiment, the cells are hematopoietic progenitor cells.
The virus and the cells are incubated in the vessel under conditions to allow the virus to infect the cells which means that the incubation is in a suitable culture medium and is for a sufficient period of time and at an appropriate temperature for infection to occur. As an example, the cells and the virus may be incubated for a period of time ranging from about 24 hours to about 48 hours at a temperature of about 37°C. The vessel can be any vessel that can be used to contain the target cells and virus including any size or shape of tissue culture ware, petri dishes, multi-well plates, membranes used in tissue culture such as hollow fiber cartridges, plastic bags, or vessel containing beads or fibres to increase the area of surface per unit volume. The vessel will comprise a material with a net positive charge which means that at least some of the surface area of the vessel will be cationic or have a positive charge. For example, the vessel can be prepared from a material with a net positive
charge or can be coated with a material that imparts a net positive charge. Preferably the vessel is coated with a cation. The cation can be any cation that imparts a positive charge to the surface of the vessel. In one embodiment, the cation is a positively charged poly-amino acid such as poly-lysine or poly-arginine, more preferable the cation is poly-L-lysine.
In a preferred embodiment, the present invention provides a method of improving viral transduction of target cells comprising:
(a) providing a vessel coated with a cation;
(b) adding a viral vector to the vessel under conditions sufficient for the vector to adhere to the vessel; and
(c) adding the target cells to be transduced to the vessel containing the vector under conditions to allow for the transduction of the cells with the viral vector.
The term "conditions sufficient for the vector to adhere to the vessel" means that the viral vector is incubated with the vessel for a period of time and at an appropriate temperature sufficient for the vector to adhere to the vessel. The viral vector is preferably incubated in step (b) on the cation coated vessel for approximately 0.1-4 hours at a temperature from about room temperature to about 37°C, more preferably 1 hour at room temperature. Preferably, the viral vector is a retroviral vector and the cation is poly-lysine.
The invention also includes a vessel coated with a cation for use in the method of the invention. The vessel coated with a cation is preferably prepared by incubating the vessel for approximately 0.1 to 4 hours at a temperature range from about room temperature to about 37°C with a cation. Preferably, the cation is poly-lysine, more preferably poly-L- lysine. The concentration of poly-lysine is preferably in the range of from about 5 μg/ml to about 100 μg/ml, more preferably about 10 μg/ml to about 50 μg/ml, most preferably 20 μg/ml, when coating standard culture dishes. One skilled in the art will appreciate that the concentration of the cation can be adjusted depending on the nature of the vessel used.
The following non-limiting examples are illustrative of the present invention:
EXAMPLES Example 1: Preparation of Cation Coated Plates Petri dishes (e.g. StemCell Technologies Inc. Vancouver,
Canada) or tissue culture dishes (Corning, Cambridge, MA, USA) are coated with 20μg/mL poly-L-lysine. Alternatively, as a control, tissue culture dishes are left uncoated, incubated with water. After 4 hours at room temperature the fluid is removed and the dishes washed with phosphate buffered saline (PBS) before adding virus containing medium (VCM). The dishes are then incubated at room temperature for another 4 hours and then the VCM removed. The dishes are washed with PBS to remove unbound virus. The cells to be transduced are suspended in culture medium with or without 5μg/mL protamine sulfate. Example 2: Transduction of K562 Cells Retroviral vectors and packaging cell lines
The retrovirus KA125, which contains the gene of the humanized redshifted green fluorescent protein (EGFP; Clontech, Palo Alto, CA) and the Neor gene, packaged in the xenotropic packaging cell line PG1329. KA125 virus produced by amphotropic GP+envAM12 cells (Markowitz, D., et al., 1988) was obtained by infecting these cells with cell-free supernatants of Bosc cells previously transfected with KA125 using CaP04 transfection (Pear et al., 1993). Transduced GP+envAM12 cells were cultured for 10 days in DMEM containing 10% bovine calf serum and 1 mg/ml G418 to select for the Neor expression and then sorted for GFP expression using a FACS Star Plus™ (Becton Dickinson, San Jose, Ca). The producer cells were shown to be free of helper virus by a helper rescue assay (Anderson et al., 1993) using RAT1 or 3T3 cells as indicator cells. Virus-containing medium (VCM) was harvested from producer cells incubated for 36-48 hours, filtered through a 0.45μm filter (Millipore,
Bedford, MA) adjusted as required to a constant titer of 3 x 105 and used either fresh or after being stored at -80 °C. Transduction protocol
Thirty-five mm petri dishes (StemCell) and tissue culture dishes (Corning) were coated as indicated with 0.5 ml H20 containing fibronectin (FN; Sigma, Oakville, ON) at 80μg/ml, 2 ml H20 containing poly-L-lysine (20 μg/ml), 2 ml H20 containing L-glutamine (20 μg/ml), or 2 ml H20 only. After 4 hours at room temperature, the fluid was removed and the dishes washed with phosphate buffered saline (PBS; StemCell) before adding 1 ml of xenotropic VCM to each dish. After 0 - 4 hours incubation at room temperature, the VCM was removed and the dishes were washed with PBS to remove unbound virus. Subsequently, 105 K562 cells were added in 1 ml IMEM plus 10% FCS in the presence or absence of protamine sulfate at 5μg/ml as indicated. For the comparison between petri dishes and tissue culture dishes, the virus loading time was adjusted to 2 hours before washing and the addition of 105 K562 cells resuspended in 1 ml IMEM plus 10% FCS with or without protamine sulfate at 5μg/ml as indicated. In some experiments, the target cells were first resuspended in VCM at 105 cells/ml and then 1ml of this mixture added to each dish. After another 48 hours at 37°C in an atmosphere of 5% C02 in air, cells were harvested and analyzed by FACS for expression of GFP. Results
The results of this experiment demonstrate the enhanced binding of infective retroviral particles to positively charged vessel surfaces. The time course and relative efficiency of retrovirus adherence to petri dishes coated with fibronectin (FN), poly-L-lysine, glutamine or uncoated are shown in Figure 1. Viral supernatants of xenotropic packaging cells producing the GFP/Neor vector adjusted to a titer of 2-4 x 105 were incubated on the various dishes for 0 to 4 hours and then the dishes were washed to remove all unbound retrovirus before adding K562
target cells (in the absence of additional virus). The percent of K562 cells found to be GFP+ 48 hours later is used and an indication of the amount of bound virus particles. Using this endpoint, very little binding of retrovirus to uncoated petri dishes or petri dishes coated with L-glutamine was demonstrated. However, Figure 2 shows a significant level of gene transfer was reproducibly seen when cells were exposed to virus that adhered to uncoated tissue culture dishes. Both dish types showed high levels of retroviral binding when coated with FN or poly-L-lysine.
High levels of retroviral transduction using FN coated petri dishes requires the presence of protamine sulfate in the medium in which the cells are incubated during transduction. Protamine sulfate does not affect the high transduction efficiencies achieved with poly-L-lysine coated petri dishes (Figure 1). The time course of the increases seen show that adherence of virus to the poly-L-lysine coated dishes is very rapid, reaching close to plateau levels within 1 hour, whereas adherence to FN was slower, requiring 2 hours to reach an equivalent level (Figure 1).
The enhanced viral binding exhibited by surfaces coated with poly-L-lysine applies to retrovirus produced by xenotrophic as well as amphotropic packaging cell lines (Figure 2). Coated petri dishes and tissue culture dishes were incubated for 2 hours with viral supernatant of PG13 cells containing the vector KA125. Unbound particles were removed. K562 cells were added with (black bars) or without (white bars) protamine sulfate and gene transfer was assessed by determining the proportion of GFP+ cells 48 hours later by FACS analysis. Coated petri dishes and tissue culture dishes were incubated for 2 hours with viral supernatant of AM12 cells containing the vector KA125. Cell addition and gene transfer assessment was performed as described above. This was not the case for uncoated tissue culture dishes which showed enhanced retroviral binding for xenotrophic virus only (Figure 2). Example 3: Transduction of Human Cord Blood Cells
Retroviral vectors and packaging cell lines were prepared as in Example 2.
Cells
Samples of cord blood (CB) from normal, full-term infants delivered by cesarean section were collected in heparin with informed consent of the mothers. Low density (<1.077g/cm3) cells were isolated by centrifugation on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) and then cryopreserved in fetal calf serum (FCS) with 10% DMSO (Sigma) at -135°C. Cord blood samples were thawed, pooled and cells expressing mature erythroid, granulopoietic, megakaryopoietic and lymphoid markers removed using a StemSep™ column (StemCell Technologies Inc., Vancouver, BC) according to the manufacturer's instructions. The resultant lineage-depleted (lήv) cell fraction contained 50 ± 7 % CD34+ cells and 2 ± 0.4 % CD34+/CD38- cells (mean ± SEM, n=6). Transduction Protocol
To infect CB cells, lin- CB cells were first incubated for 48 hours in Iscove's medium supplemented with a serum substitute (BIT™, StemCell Technologies Inc, Vancouver, Canada), 10"4 M 2-mercaptoethanol (Sigma) and the following human recombinant growth factors: flt3-ligand (FL, lOOμg/ml, Immunex Corp, Seattle, WA), Steel factor
(SF, lOOμg/ml, prepared from cDNA transfected Cos cells in the Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia), IL-3 (20μg/ml, Novartis, Basel, Switzerland), IL-6 (20μg/ml, Cangene, Mississauga, ON) and G-CSF (20μg/ml, StemCell). The cells were then centrifuged and resuspended in virus containing medium (VCM) to which protamine sulfate (5μg/ml, Sigma) and the same cytokines were also added. One ml containing 105 cells were then added to petri dishes preloaded twice with VCM (30 min each time) for 24 hours. The cells were then harvested resuspended in new VCM plus protamine sulfate and cytokines and added to new preloaded dishes. At the end of the third infection, the cells were left in the dishes for another 24 hours and then harvested. FACS analysis was used to determine the proportion of GFP+ CD34+ cells as described (Hennemann, B. et al., 1999). In addition, some
cells were plated in methylcellulose medium (HC4334, StemCell) with and without 1.7 mg G418 (dry powder weight) to determine the proportion of G418 resistant colony-forming cells (CFC) (Hennemann, B. et al., 1999). Other transduced cells were injected intravenously into sublethally irradiated (350cGy) NOD/SCID mice together with irradiated human marrow carrier cells (Hennemann, B. et al., 1999). Gene transfer to NOD/SCID mouse repopulating cells was calculated by dividing the number of GFP+ human CD45+ cells by the total number of human CD45+ cells found to be present 6 to 8 weeks later in the BM of mice showing engraftment with both human lymphoid and myeloid cells (defined by the presence of both >5 human CD19/20+/CD34" and >5 human CD15+ cells per 20,000 cells) (Holyoake, T.L. et al., 1999). Results
The results of this example demonstrate high efficiency retroviral transduction of primary human cord blood cells in tissue culture dishes without FN.
The utility of tissue culture dishes for the retroviral transduction of primary human hematopoietic stem cells was compared to FN-coated dishes. As described, in the Experimental protocol, l v cord blood cells were therefore infected for 3 times on tissue culture dishes or FN-coated petri dishes preloaded for 2 hours with PG13-KA125 supernatants (3x105 U/ml) and the efficiency of gene transfer to CD34+ cells, CFC, LTC-IC and NOD/SCID repopulating cells was assessed 48 hours after exposure to new virus for the third time. As shown in Table 1, both the percent and yield of transduced cells were the same for all cell types measured in vitro for both arms of the experiment. Gene transfer efficiencies to transplantable stem cells assessed by measuring the expression of GFP in the lymphoid (CD19/20+/CD34-) and myeloid (CD15+) progeny as well as expression of GFP in all human cells (human CD45+ or human CD71+) they produced in engrafted NOD/SCID mice as shown in Figure 3. Figure 3 shows FACs profiles of bone marrow cells obtained from a mouse transplanted with transduced human CB cells 6 weeks
previously and then stained with various anti-human monoclonal antibodies. Human liir CB cells were transduced once daily for each of three days in uncoated tissue culture dishes loaded for 2 hours with viral supernatant of PG13 cells. The combined results of 5 experiments are shown in Figure 4, where it can be seen that the proportion of regenerated GFP+ human cells was higher, although not yet significantly (pθ.08) in NOD/SCID recipients of cells that had been transduced in tissue culture dishes (mean ± SEM = 15 ± 6 %) as compared to FN-coated petri dishes (4 ± 1) (Panel B). The level of engraftment achieved by both sources of cells (Panel A) as well as the calculation of the overall yield of transduced cells obtained in the mice after injection of equivalent proportions of the original cells infected showed no difference (p=0.9 and p=0.5, Panel C). DISCUSSION
The stable insertion of new genetic material into primitive hematopoietic cells capable of long- and short-term reconstitution of mature blood cells remains a crucial step for the use of the retroviral technique in both the study of hematopoiesis and the treatment of diseases of the blood and the immune system. The low gene transfer rates to hematopoietic cells obtained so far are thought to be caused in part by the low rate of successful virus /target cell encounters, including low virus titers obtained with large virus constructs (Ward, M. et al., 1994; Hanania, E. G. et al., 1996) and the short half-life of infective retroviruses (Kotani, H. et al., 1994; Chuck, A. S. and Palsson, B. 0. 1996).
With the present invention, this problem has been overcome. The initial absorption of retroviruses onto the surface of target cells occurs randomly due to Brownian motion and gravitational settling which then allows the specific high affinity binding of viruses to their cellular receptors (Weiss, R. A. and Tailor, C. S. 1995; Palsson, B. and Andreadis, S. 1997). Because both the outer cell membrane and the retrovirus envelope are predominantly negatively charged (Toyoshima, K. and Vogt, P. K. 1969; Darnell, J., Lodish, H. and Baltimore, D. 1986), repelling electrostatic forces can be postulated to reduce the frequency of successful virus target cell
encounters. In addition, the viability of retroviral particles is time limited, and therefore the distance functional particles can travel until absorbed onto a cell is also limited. Hence, the method of the invention that increases the frequency of collisions between viruses and cells will improve gene transfer efficiencies up to the point where other factors become limiting.
Retroviral particles do bind directly and with high efficiency to plastic surfaces coated with a cationic amino acid and, to a lesser extent, to FN-coated petri dishes or positively charged, uncoated tissue culture dishes.
Although polycations were shown to enhance the infection efficiencies of a number of viruses, the proposed mechanism of action is the generation of a cell/polycation/virus particle sandwich, in which the polycation mediates apposition of the negatively charged cell and viral surface membranes resulting in an improvement in the efficiency of uptake of virions (Toyoshima, K. and Vogt, P. K., 1969; Ryser, H. J., 1967; Manning, J. S. et al., 1971). This hypothesis led to the widespread use of polycations such as polybrene and protamine sulfate in gene transfer protocols although no one proposed immobilizing the polycations or a positive charge on a surface to facilitate gene transfer by viral vectors. The results of the present invention show that it is not necessary for the cationic mediator to be in a position between the target cell and retroviral particle if the retrovirus is stuck onto a cationic surface. While not wishing to be bound by a particular theory, two mechanisms by which surface bound cationic charges may increase the transduction efficiencies include: the reduction of negative charges on the outer membrane of the retroviral envelope by neutralization and thereby the alleviation of repellent electrostatic forces; and the fixation of the retrovirus particle on the surface by electrostatic binding and thus reducing the repulsing effect of equipolar charges on the larger cell.
The number of infective virus particles reaching a susceptible target cell is increased by prolonged preloading of suitable positively
charged surfaces. Because it has previously been reported that retroviral particles can be concentrated by repeated exposure of CH-296 coated bacterial petri dishes (Hanenberg, H. et al., 1997), the enriching effect achieved on tissue culture treated polystyrene was compared to the one achieved on FN coated bacterial petri dishes. The mechanism of action of FN or CH-296 has been described as being mediated by specific binding of the retrovirus to repeats 12-14 of FN, which are adjacent to repeats 8-9 and the CS1 binding site of the FN or CH-296-molecule to which the target cells specifically bind via VLA-5 and VLA-4 as ligands (Hanenberg, H. et al., 1996; Hanenberg, H. et al., 1997). Hence, it was postulated that the gene transfer efficiency is reduced to levels achieved by supernatant alone if the binding sites for either retrovirus or target cells are missing (Hanenberg, H. et al., 1996; Hanenberg, H. et al., 1997). The present results show that by co-localization on positively charged polystyrene also target cells of various species not expressing VLA-5 or VLA-4 can be efficiently transduced. This observation significantly exceeds the results obtained on FN or FN fragments. Simple co-localization of retrovirus and target cells on positively charged or tissue culture treated plastic widens the range of potential target cells and provides a tool to address and improve retroviral transduction of virtually every cell type in a given population of susceptible cells.
The feasibility of this approach has been demonstrated by targeting primary hematopoietic progenitor and multi-lineage in vivo repopulating stem cells in human lineage depleted umbilical cord blood. The gene transfer efficiency as well as cell yield of CD34+ cells, CFC (colony forming cells) and LTC-IC (Long term culture - initiating cells) are similar with transduction on uncoated positively charged tissue culture dishes or on FN-coated petri dishes. Transduction of multi-lineage in vivo repopulating cells is higher on the uncoated tissue culture dishes. This proves that fibronectin is not necessary for the transduction of in vivo repopulating cells and the transfer of therapeutical genes into
hematopoietic cells need not involve exposure to human pathogens potentially present in human plasma derived fibronectin.
While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
TABLE 1
Comparison of Gene Transfer Efficiencies and Yield of Genetically Modified Primary Hematopoietic Cells Using Tissue Culture Dishes vs
FN-coated Petri Dishes
Infection Transduction Efficiency Yield Container (%) (Fold Expansion)
CD34+ CFC LTC-IC CD34+ CFC LTC-IC
Petri Dish+ FN 57 ± 8 62 ± 10 77 ± 23 2.1 ± 0.7 3.3 + 1.4 3.3 ± 1.1
Tissue Culture 59 ± 7.0 62 ± 3 54 ± 27 2.0 ± 0.6 3.4 ± 1.3 4.7 ± 1.7
Dish
Lin- CB cells were stimulated for 2 days with FL + SF + IL-3 + IL-6 + G-CSF and then exposed to new virus daily for 3 days. Two days after the last exposure to fresh virus, the cells were harvested and either analyzed by FACS for GFP expression on CD34+ cells or tested in functional assays to determine the proportion of G418-resistant CFC and LTC-IC. All yields are expressed as fold increases relative to the number of the corresponding cells type initially placed in culture. Data for CD34+ cells and CFC are given as mean ± SEM from 6 individual experiments, and for LTC-IC from 3 experiments.
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