DE19900284B4 - Method for the efficient introduction of DNA into eukaryotic cells using an adenoviral vector - Google Patents

Abstract

method for introducing DNA into eukaryotic cells, characterized that adenoviruses that contain the DNA to be introduced, with liposomes are pre-incubated and subsequently with eukaryotic cells be contacted, the preincubation for about 30 Minutes at about 37 ° C is carried out, where the eukaryotic cells are not human embryonic Stem cells act.

Description

The The present invention relates to a method for introducing DNA (deoxyribonucleic acid) in Eukaryotic cells, especially lymphocytes, using a Adenoviral vector and a method of producing a therapeutic effective cell preparation using the above method.

Of the Transfer of genetic information to eukaryotic cells is one of the Most-processed work areas of modern biomedical Research. Especially in the field of gene therapy hang more Progress to a high degree from the development of suitable systems for the introduction of exogenous DNA in target cells, since gene therapy approaches both an efficient Transfer of genes as well as correct expression of the gene products in the target cells. There are therefore many methods have been developed for gene transfer to eukaryotic cells.

Some These methods are based on the transfection of target cells under Use of liposomes. However, these methods have the disadvantage on that that Spectrum of the transfected cells is limited. Thus, e.g. Lymphocytes, the most promising target cells for gene therapy approaches not sufficiently efficient according to these methods be transfected. Similar restrictions also apply to Procedures based on the transfection of eukaryotic cells by means of Based electroporation.

Because of their highly efficient gene transfer to eukaryotic cells have become Vector systems based on viruses are particularly promising proved. A big part These systems rely on retroviruses that provide high transfer efficiency but infect only actively dividing cells.

adenoviral Vectors offer some potential advantages over retroviral ones Vectors, in particular for the immunomodulation of lymphoma cells. Several publications are coming but to the conclusion that humane compared to lymphoid cells adenoviral infections are resistant (Prince HM, Dessureault S. Gallinger S, Krajden M, Sutherland DR, Addison C. Zhang Y, Graham FL, Stewart AK: Efficient adenovirus-mediated gene expression in malignant human plasma cells: relative lymphoid cell resistance. Exp. Hematol. 26:27, 1998; Cantwell MJ, Sharma S, Friedmann T. Kipps TJ: adenovirus vector infection of chronic lymphocytic leukemia B cells. Blond 88: 4676, 1996; Silver L, Anderson CW: Interaction of human adenovirus serotype 2 with human lymphoid cells. Virology 165: 377, 1988). Furthermore several authors come to the conclusion that adenoviral vectors by their suboptimal efficiency and their high toxicity Concentrations for the transfer of genetic information to target cells of limited benefit (Rosenfeld MA, Yoshimura K, Trapnell BC, Yoneyama K, Rosenthal ER, Dalemans W, Fukayama M, Bargon J, Taurus LE, Stratford Perricaudet L, et al .: In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium. Cell 68: 143, 1992; Rystal RF, McElvaney NG, Rosenfeld MA, Chu CS, Mastrangeli A, Hay JG, Brody SL, Jaffe HA, Eissa NT, Danel C: Administration of adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat Genet 8:42, 1994; Knowles MR, Hohneker KW, Zhou Z, Olsen JC, Noah TL, Hu PC, Leigh MW, Engelhardt JF, Edwards LJ, Jones KR, et al .: A controlled study of adenoviral vector-mediated gene transfer into the nasal epithelium of patients with cystic fibrosis. N Engl J Med 333: 823, 1995; Schulick AH, Newman KD, Virmani R, Dichek DA: In vivo gene transfer into injured carotid arteries. Optimization and evaluation of acute toxicity. Circulation 91: 2407, 1995). Prince et al. (Prince HM, Dessureault S. Gallinger S, Krajden M, Sutherland DR, Addison C. Zhang Y, Graham FL, Stewart AK: Efficient adenovirus-mediated gene expression in malignant human plasma cells: relative lymphoid cell resistance. Exp. Hematol. 26:27, 1998) report transfection efficiency for B lymphocytes of only 14%. The expression of LacZ or luciferase was in lymphoma cells treated by the transfection method almost 100 times lower than in appropriately treated OCI-My5 myeloma cells, even if very high multiplicities the infection was used.

The WO 98/46781 describes an expression system comprising a transcriptional unit that contains a transgene and is introduced into a cell along with a portion or all of the adenovirus E4 gene region to allow for persistent expression of the transgene.

The WO 98/50053 relates to the use of a modified adenovirus as a gene delivery vehicle. The US 5,543,328 describes an adenovirus as a gene delivery vehicle modified to have a ligand that allows for targeting to a desired cell type.

Qiu C., et al. ("Cationic liposomes enhance adenovirus entry via a pathway independent of the fiber receptor and alpha (v) integrins ", in: Hum Gene Ther. 1998 Mar 1; 9 (4): 507-20) describes that the ability of adenoviral sectors to allow efficient gene transfer both in vitro and in vivo Furthermore, it is stated that the use of liposomes has not led to an improvement in the adenoviral mediated gene transfer to important target cells.

An overview of common transfection procedures is found in "Human Cancer and Gene Therapy in Ann. Hematol. (1994) 69, 273-279. The There described methods have the disadvantages mentioned above on.

As a result, The present invention is based on the object, a method provide high-efficiency DNA into a wide range of DNA Spectrum of eukaryotic cells can be introduced and a propose advantageous use of the processed products.

According to the invention this Task first solved by a method which is characterized in that Adenviren, the to be inserted DNA are pre-incubated with liposomes and then with Eukaryotic cells are contacted, with the preincubation for about 30 minutes at about 37 ° C carried out , wherein the eukaryotic cells are not human embryonic stem cells. The solution of the above task is done also by using the modified obtained by the above method Eukaryotic cells for the production of a therapeutically effective Cell preparation.

The Spectrum of eukaryotic cells produced by the method of the present invention Invention can be modified is not subject to any fundamental restrictions. Preferred target cells are mammalian cells, especially human cells. With regard to gene therapy applications are preferably lymphocytes of interest. Especially preferred Cells are lymphoma cells, especially B lymphoma cells, which are interesting Starting points for Gene therapy concepts for the treatment of cancers represent, i.a. due to the fact that enlarged lymph nodes easily accessible are and these cells for immunological strategies appear appropriate. Thus, e.g. many lymphomas on treatment with interferon α. EBV (Epstein-Barr virus) based vectors can used for targeted gene transfer to EBV-associated neoplasms become. In another approach could be shown that the targeted Destruction of the beta-1 integrin gene in a lymphoma cell line's ability this, to form metastases, greatly reduced. lymphoma cells are resistant to Most recent methods of gene transfer that prove to be too toxic to these cells have proven. In particular, the methods of transfection by electroporation or with the aid of liposomes in lymphoma cells largely ineffective.

By the inventive method DNA is introduced into eukaryotic cells. Here is the infiltration of DNA comprising one or more genes of preferred interest. Preferably, the DNA in addition regulatory elements. Regulatory elements are promoters, Polyadenylation signals and / or origins of replication, in particular emphasized. With regard to gene therapy applications is the transfer of DNA which expresses the expression of the corresponding gene product in the target cells result. For the expressed gene product it is preferably a cytokine. Especially preferred Cytokines are, especially in the context of gene therapy approaches on lymphoma cells, the interleukins IL-2, IL-7 and IL-12.

It but also the possibility considered to infiltrate DNA into cells for expression to suppress another gene. These preferably include so-called "anti-sense" genes which encode an "antisense" RNA or a ribozyme. In a preferred embodiment For example, the "antisense" RNA is a bcl-2 "anti-sense" RNA, i. one RNA that hinders or prevents the expression of the bcl-2 gene.

The method according to the invention preferably comprises the incubation of the eukaryotic cells with adenoviruses containing the DNA to be injected, the incubation taking place in an incubation buffer. The incubation buffer is preferably PBS (phosphate buffered saline, 10 mmol / l sodium / potassium phosphate buffer pH 7.2, 0.8% NaCl and 0.02% KCl) + 1 mmol / l MgCl ₂ /1%. Horse serum (hereafter HS, for horse serum). An important experimental parameter is the numerical ratio of viruses to cells. This numerical ratio is expressed by the multiplicity of infection (hereinafter referred to as MOI, called multiplicity of infection), which is defined as the quotient of the number of viruses used and the number of cells to be infected. In a preferred embodiment of the present invention, the MOI is about 1 to 200.

From Of crucial importance is also the period of time over which the incubation of the eukaryotic cells with the adenoviruses takes place and a low incubation volume. Long incubation times increase In general, the efficiency of the transfer of genetic information and the low volume increases the number of hits of adenoviruses on the cell. simultaneously However, it also increases the risk of toxic effects on the cells. The inventive method allows long incubation times without compromising the viability of the target cells impaired becomes. Preferably, after about 2 hours of incubation, for example at about 37 ° C, the target cells with the adenoviruses, the DNA to be injected included, complete Cell culture medium was added and the incubation then for about 1 continued until 14 days.

As said above leads the transfection of eukaryotic cells with the help of liposomes often to unsatisfactory results, especially when it comes to the Transfer of genetic information to lymphocytes goes. Surprisingly has been shown that the described method leads to particularly good results, if it is combined with treatment by liposomes. Therefore, be the adenoviruses before incubation with the eukaryotic cells Pre-incubated liposomes. This preincubation takes place for about 30 Minutes at about 37 ° C instead of.

The inventive method is suitable to obtain eukaryotic cells by transfer of genetic Information has been modified. Preference is given to eukaryotic cells obtained in which the introduced foreign genes are expressed. By the method according to the invention will it be possible to produce a therapeutically effective cell preparation by taken from a population of cells of a mammal and after the inventive method is modified.

The present method is suitable for the transfer of genetic information on a wide range of eukaryotic cells, especially lymphocytes. It was found that a transferred gene expressed in up to 100% of the treated cells was, without the so treated cells in their growth properties on a larger scale impaired have been. Across from Retroviruses whose use is restricted to actively dividing cells, The present adenoviral system offers the advantage that it too is applicable to non-actively dividing cells. Another The advantage of adenoviruses is that they produce slightly high titers can be obtained. The fact that through Adenoviruses transferred DNA is apparently not integrated into the genome of the target cell and the resulting gene expression is therefore transient (non-persistent) is, is for many gene therapy approaches without disadvantages or even an advantage.

In summary let yourself say that the main advantages of the method according to the invention in the high Efficiency of gene transfer, applicability to a wide range of eukaryotic cells and ease of operation.

The following examples serve to understand the following examples:
In the examples, the invention for the transfer of genetic information with conventional methods, ie transfection by electroporation and transfection with the aid of liposomes, compared. The target cells were the cell lines Raji (human Burkitt lymphoma cell line, obtained from DSMZ, Braunschweig, Germany), Daudi (human Burkitt lymphoma cell line, obtained from DSMZ, Braunschweig, Germany), OCI-Ly8-LAM53 (R. Levy, Stanford University, USA) and SU-DHL-4 (B lymphoma cell line, R. Levy, Stanford University, USA). The cell lines were analyzed in the medium RPMI 1640 with Glutamax (GIBCO, Berlin, Germany) supplemented with 10% heat-inactivated fetal calf serum (FCS (PAA, Martinsried, Germany)) and with 50 μg / ml penicillin in a humid incubator with 5% CO ₂ cultured at 37 ° C.

The Propagation of the adenovirus was carried out in the Ad5 E1-transformed human embryonic retinal cell line 911 (Fallaux FJ, Kranenburg O, Cramer SJ, Houweling A, Van Ormondt H, Hoeben RC, Van Der Eb AJ: Characterization of 911: a new cell line for the titration and propagation of early region 1-deleted adenoviral vectors. Hum Gene Ther 7: 215, 1996). This cell line was modified in Dulbecco's Eagle Medium (DMEM, GIBCO) cultured with 10% FCS, 50 μg / ml streptomycin and 50 μg / ml Penicillin was supplemented.

As the gene to be transferred, β-gal was selected, which allows a simple determination of the efficiency of the transfer, since its gene product is incubated with incubation of the treated cells with X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside , β-gal staining kit, Invitrogen, de Schelp, The Netherlands) leads to a clearly visible and evaluable blue coloration. For evaluation at least 400 cells were counted after each experiment. The plasmids used in the experiments are described in Table 1. Table 1 vector company characteristics reference pcDNA3.1 / HisB / LacZ Invitrogen (Netherlands) 8578bp; CMV promoter; LacZ ORF, BGH polyadenylation signal Invitrogen (Netherlands) Ad-β-Gal K. Brand (Humboldt University, Berlin, Germany) E1 and E3 deleted replication defective adenovirus type 5, E. coli LacZ gene under the control of a CMV promoter K. Brand (Sandig v, Brand K. Herwig S, Lukas J, Bartek J, Strauss M: Adenovirally transferred p16INK4 / CDKN2 and p53 genes cooperate to induce apoptotic tumor cell death., Nat Med 3: 313, 1997

The Invention will be explained in more detail by the following examples 1 to 5.

example 1

Transfection by electroporation

Raji and Daudi cells were prepared using the electroporation EASYJECT plus ^® from Eurogentec, Seraing, Belgium, transfected with the plasmid pcDNA3.1 / HisB / LacZ. For this 5 × 10 ⁶ cells were incubated in 500 μl complete RPMI 1640 medium and mixed with 30 μg pcDNA3.1 / HisB / LacZ. This mixture was transferred to 4 mm electroporation cuvettes, incubated on ice for 10 minutes and pulsed with a single or double pulse program. The parameters for the first pulse were 250 V and 1650 to 2100 μF. After the pulse, the cells were transferred at a density of 1 x 10 ⁶ cells per ml in complete RPMI 1640 medium. The transfection efficiency was determined 24 and 48 hours after transfection.

1 shows that only a very low transfection efficiency was achieved by this method. It was less than 2% for both Daudi and Raji cells. Viability of the cells was not affected by electroporation (data not shown).

Example 2

Transfer by lipofection

Raji and Daudi cells were transfected with pcDNA3.1 / HisB / LacZ using various liposomal or liposome-like reagents (Table 2). The transfections were carried out as described in the respective manuals of the reagents. The cells were seeded in 24-well plates at densities between 0.5 to 5 x 10 ⁵ cells per ml in RPMI 1640 with or without FCS (as described in the respective manuals). The DNA / transfection reagent mixtures were prepared by first separately diluting a suitable amount of the reagent (0.1 to 10 μl) and the DNA (0.1 to 5 μg) in serum-free Opti-MEM (GIBCO, Berlin, Germany) or RPMI 1640 has been. Subsequently, the transfection solution was added to the DNA solution and incubated at 25 ° C for 10 to 30 minutes. After formation of the transfection reagent / DNA complex, it was added dropwise to the cells. The cells were incubated for 2 to 24 hours at 37 ° C in 5% CO ₂ as described in the respective manuals. In some cases it was not necessary to remove the transfection complexes. After 24 to 48 hours, cells were stained for β-gal expression. The results are summarized in Table 2. Table 2 Transfection / method characteristics cell line Efficiency (percentage of cells expressing β-galatosidase) Superfect ^® (Qiagen, Hilden, Germany) non-liposomal hyperbranched polycationic transfection reagent Daudi: Raji: <1% <1% Effectene ^® (Qiagen, Hilden, Germany) non-liposomal lipid uses a specific DNA condensing enhancer Daudi: Raji: <1% 1-3.5% Dosper ^® (Boehringer Mannheim, Mannheim, Germany) polycationic lipid (1,3-dioleoyl-oxy-2- (6-carboxyspermyl) -propylamide) Daudi: Raji: <1% <1% Fugene 6 ^® (Boehringer Mannheim, Mannheim, Germany) non-liposomal preparation Daudi: Raji: <1% <1% Lipofectamine ^® (Gibco-Life Technologies, Karlsruhe, Germany) 3: 1 formulation of a polycationic lipid (DOSPA) and a neutral lipid (dope) Daudi: Raji: <1% 5-10% DAC-30 ^® (Eurogentec, Seraing, Belgium) liposomal formulation of a monocationic cholesterol derivative Daudi: Raji: 0% 0%

Comparison of different liposomal Transfection reagents with respect to the transfection efficiency of Daudi and Rajicella

The transfection efficiency was both Daudi and for Rajizellen below 1%, except for Effectene ^® and lipofectamine ^®, the transfection efficiencies of up to 10% showed at Rajizellen.

Example 3

Adenoviral gene transfer (method according to the invention)

The recombinant adenoviral vector Ad-β-Gal carries the E. coli LacZ gene encoding β-galactosidase (β-Gal) under the control of the promoter of the Rous sarcoma virus. Ad-β-gal were obtained from Dr. Karsten Brand provided. Plaque essays were performed essentially as described by Graham and Prevec (Graham F, Prevec L: Manipulation of Adenovirus Vectors, Methods in Molecular Biology 7: Gene Transfer and Expression Protocols, 109, 1991). Adenovirus stocks were serially diluted in 2 ml DMEM (GIBCO) containing 2% horse serum (HS) and added to nearly confluent 911 cells in 6-well plates. After 2 hours incubation at room temperature (25 ° C), the medium was replaced by F-15 minimal essential medium (MEM, GIBCO) containing 1% agarose (Sigma, Deisenhofen, Germany), 20 mmol / l Hepes pH 7, 4, 0.0025% L-glutamine, 5% yeast extract, 8.4% NaHCO ₃ , 50 μg / ml streptomycin and 50 μg / ml penicillin and 2% HS. The plaque titer of Ad-β-gal was 3.5 × 10 ⁹ pfu / ml (pfu stands for plaque-forming units). The production of adenovirus lots was performed as described by Fallaux et al. Described by the authors of the case study (Fallaux FJ, Kranenburg O, Cramer SJ, Houweling A, Van Ormondt H, Hoeben RC, Van Der Eb AJ: Characterization of 911: a new helper cell line for the titration and propagation of early-1-deleted adenoviral vectors Gene Ther 7: 215, 1996). For this, nearly confluent 911 monolayers in 175 cm ² flasks in 2 ml PBS containing 1% HS were infected with 5 pfu per cell. After 1 hour at room temperature, the inoculum was replaced with fresh medium (DMEM / 2% HS). After 48 hours, almost completely detached cells were harvested and taken up in 1 ml PBS / 1% HS. The virus was isolated by three cycles of rapid deep freezing and thawing. The lysates were by Zen Purification at 3000 rev / min for 10 minutes clarified. The virus stock lots were stored in PBS / 10% glycerol at -80 ° C. Infection of the cells was carried out at 5 x 10 ⁵ cells in 50 μl PBS + 1 mmol / l MgCl ₂ /1% HS, at different MOI (0 to 200), with Ad-β-gal. After incubation for 2 hours at 37 ° C, the infected cells were given 1 ml of complete culture medium (a medium containing, besides the essential compounds, also many compounds which the organism can synthesize itself, thereby increasing the growth rate, for example, RPMI 1640 + glutamax + 10% heat-inactivated FCS). Since no visible toxic effect was observed in comparison to the controls (PBS + 1 mmol / l MgCl ₂ /1% HS only), it was not necessary to remove the viruses.

2 shows that this method succeeded in an efficient transfer of the genetic information to the cells.

The Transfection efficiency was dose-dependent. At an MOI of 200 100% of both Daudi and Rajic were 48 and Transfected for 72 hours. The Daudi and Rajic cells were added to the Ad-β-Gal for 1 to 14 days suspended. X-gal staining showed 100% infected cells after 72 hours.

Example 4

Adenoviral gene transfer with the aid of of liposomes

The experiments were performed as described in Example 3, but Ad-β-Gal was also before the infection of the target cells with Lipofectamine ^® (GIBCO) at a final concentration of 20 ug / ml in 50 ul Opti-MEM pre-incubated. The virus-lipid mixture was incubated for 30 minutes at 37 ° C before being added to the target cells.

3 shows that by this method, the transfection efficiency could be increased again.

Compared to the procedure without liposomes, the use of cationic liposomes resulted in more transfected cells upon infection with lower concentrations of the adenovirus. At lower vector concentrations (MOI 50), the liposomes increased transfection efficiency ( 3 ). At higher vector concentrations, the improvements achieved by liposomes were lower.

Example 5

Growth characteristics after transfection

In order to investigate a possible impairment of the growth behavior of the infected cells, their growth behavior was compared with the uninfected cells. The viability determination was carried out by trypan blue exclusion. The comparison cells used were cells which had been treated with empty incubation buffer (PBS + 1 mmol / l MgCl ₂ /1% HS). Uninfected and Ad-β-gal infected cells were counted after infection for up to 14 days every 24 hours.

4 shows that the infected cells were not affected in their growth. Cell growth after adenoviral transfection with an MOI of up to 200 was not significantly different from untransfected cells.

Claims (15)

A method for introducing DNA into eukaryotic cells, characterized in that adenoviruses containing the DNA to be introduced are preincubated with liposomes and then contacted with eukaryotic cells, wherein the preincubation is carried out for about 30 minutes at about 37 ° C the eukaryotic cells are not human embryonic stem cells. Method according to claim 1, characterized in that that the eukaryotic cells are mammalian cells, especially human cells. Method according to claim 1 or 2, characterized that the eukaryotic cells are lymphocytes. Method according to one of claims 1 to 3, characterized that the eukaryotic cells are lymphoma cells, in particular B-lymphoma cells, are. Method according to one of claims 1 to 4, characterized that the DNA comprises one or more genes. Method according to claim 5, characterized in that that the DNA in addition includes regulatory elements. Method according to claim 5 or 6, characterized that it involves the expression of the gene product. Method according to claim 7, characterized in that that the gene product is a cytokine. Method according to claim 8, characterized in that that it the cytokine is IL-2, IL-7 or IL-12. Method according to one of claims 1 to 6, characterized that it involves transcription to an anti-sense RNA. Method according to claim 10, characterized in that that it the "anti-sense" RNA is bcl-2 "anti-sense" RNA. Method according to at least one of the preceding claims, characterized in that it involves the incubation of the eukaryotic cells with adenoviruses containing the DNA to be injected, the incubation taking place in the following incubation buffer: PBS + 1 mmol / l MgCl ₂ /1% HS Method according to at least one of the preceding Claims, characterized in that a multiplicity of infection (MOI) of about 1 to 200 is used. Method according to at least one of the preceding Claims, characterized in that after about 2 hours incubation complete Cell culture medium is added and then the incubation for about 1 continues until 14 days. Use of according to a procedure after at least one of the claims 1 to 15 modified eukaryotic cells obtained for the preparation a therapeutically effective cell preparation.