DE10015906B4 - Directed gene transfer into Thy-1 positive cells - Google Patents

Abstract

Method for directed gene transfer into human fibroblastoid cells, characterized in that
Polyethyleneimine with a molecular weight <2,000 kDa
with a plasmid containing the tissue-specific promoter Thy-1 or a nuclear localization signal,
is implemented in such a way
the ratio of the nitrogen of the polyethyleneimine to the phosphor of the nucleic acid is 2: 1 to 48: 1,
the resulting construct is reacted with a monoclonal antibody to recognize human fibroblasts such
that the linking takes place with modification of the sugar residue of the monoclonal antibody or its amino group,
the conjugate is pegylated and
used for gene transfer.

Description

The The invention relates to the field of gene transfer to certain eukaryotic cells.

you means by gene transfer the introduction of one or more Genes in cells to cause a genetic defect either in these cells correct or control the cells certain gene products otherwise they would not be in this lot or at all do not synthesize. These include in addition to indicators (e.g., fluorescent dyes) growth factors, Cytokines, hormones, but also enzymes. This type of gene transfer can either Modification of cells in culture or clinical gene therapy with change of somatic cells to the target. The change of germ line cells with the goal of transgenic organisms, however, belongs technically and legally Do not dictate this type of gene transfer.

Around To make the application of somatic gene transfer efficient must have different preconditions Fulfills be.

The Hang prerequisites This also depends on the type of transfer system used. Basically investigated viral and non-viral transfer systems for several years (Blaese et al., 1995), with viral systems so far favoring more were. Due to lethal side effects due to overstimulation of the immune system at high doses of recombinant virus particles after gene therapy in younger Time viral procedures are considered problematic. Also at low doses of recombinant virus is repeated when given with a weakening the effect to be expected because the recipient against this recombinant Viruses form antibodies becomes. The advantage of viral systems (Smith, 1995), especially of adenoviruses (Trapnell and Gorziglia, 1994) and adeno-associated virus (AAV; Russell et al., 1994) is high in its, but sometimes in vivo relatively unselective transfection efficiency. An increase in the selectivity succeeds through the use of different, system-adapted vectors, in particular those of retroviruses (Schnierle and Groner, 1996; Mouse Mammary Tumor virus, MMTV; Lentiviruses such as HIV). Own viruses generally a host cell-specific tropism. In vivo shows up however, that the Specificity not is the same in all systems. In addition to the risk of immunological Side effects also exist the possibility of recombination of viral genomic parts of the vectors leading to pathological changes of the genome in cells of the recipient to lead can. Newer scientific assessments assume that at least 1% of the genome of higher mammals integrated retroviral components are or similar proto-oncogenes. Therefore, the probability of unwanted recombination effects quite as not very low estimate.

Therefore Some new developments aim to move to non-viral systems, and doing properties of the virus systems that are relatively good Transfection efficiency, if possible (Coonrod et al., 1997; Mahato et al., 1997).

Accordingly, the requirements for effective non-viral gene delivery systems, in particular for in vivo therapies, but not exclusively, can be summarized as follows:
They need to be able to stay intact in the blood stream for a while while being small enough to enter capillaries, tissues and cells.

Further have to they may be protected from processing in endosomes / lysosomal complexes, thus the DNA into the nucleus of the host cell for subsequent transcription the genes arrive.

As a general rule desirable is finally a flexible tropism, about the vectors can be as specific as possible "addressed" to different target cells or diseases.

Necessary are cell-specific promoters, about which for certain cell types the optimal Transcription is controlled.

The previously available Non-viral gene transfer systems try to address some of these needs to fulfill, However, they are unable to target certain human connective tissue cells like fibroblasts or mesenchymal stem cells as their precursor as well efficiently transfecting some human neuronal cells (examples). Compared to standardized, mostly transformed cell lines (COS-1, COS-7, HeLa, K-562, HepG2 et al.) Allows gene transfer into primary human Fibroblasts with the previously available Perform funds only with very low efficiency.

Accordingly, the object of the invention is to circumvent the dangers of using (retro) viral vectors by a non-viral system and, as far as possible, to achieve the efficiency and reliability of viral systems. In particular, the system according to the invention should be able to specifically carry out gene transfer into human Thy-1-positive cells. For this purpose, a Thy-1-specific monoclonal antibody is coupled to a polycation via a bridge. With the help of the Thy-1 specific Antibody is the specific addressing of Thy-1-positive human cells allows, are transferred to the integrated polycation condensed nucleic acids.

In DE 197 26 186 For example, antibodies are mentioned to describe the functionality of the gene delivery system, but in the examples only transferrin that is not an antibody is used. However, as transferrin is almost universally expressed on mammalian cells, the construct diminishes DE 197 26 186 the opposite of a goal-directed gene transfer based on a cell-specific seeker.

Ansell S. M. et al Bioconjug.Chem. 1996 Jul-Aug: 7 (4): 490-6 describe 3- (2-pyridildithio) propionic acid hydrazide as a cross-linker for the formation of liposome-antibody conjugates. With the given information, however, the task can be left not solve the invention in question here, because the coupling of antibodies indicated linker to specific fibroblast antibodies, polyethyleneimine, Polyethylene glycol and a corresponding Thy-1 promoter containing Do not suggest the plasmid.

to Complexation of nucleic acids various polycations are used, such as polylysine (Wu and Wu, 1987), Protamine, spermine (Kichler et al., 1995) or polyethylenimine (PEI) successfully used (Boussif et al., 1995; 1996; Abdallah et al. 1996, Kircheis et al., 1997). Cells take such complexes, which bind undirected to the cell membrane. To the complexes specificity various ligands such as asialoglycoportein, transferrin, Mannose, furosis, antibodies, Asialofuetin, vascular Endothelial growth factor, fibroblast growth factor, cholera toxin B or folic acid used, which were covalently coupled to the polycation. So far The best use was of the transferrin receptor for gene transfer examined. All active metabolizing cells need iron, that is endocytosed by the cells as a transferrin-iron complex becomes (Kircheis et al., 1997). This commonly used recording mechanism, but from the outset a restriction to very specific cell types excludes was also for the uptake of nucleic acids, Proteins and other molecules examined. Complexes of transferrin and polylysine, protamine or PEI were used to express the gene for the enzyme luciferase in cells bring to. First, it was chicken erythroblasts, which were transformed with the proto-oncogene erbB because known is that this Cells have a particularly active transferrin metabolism, for other the human leukemia line K562, which has relatively many transferrin receptors on the cell surface. With these two cell lines, an effective transfection was accomplished which is a number of 10-20 transferrin molecules per Complex as optimal for the transfection has exposed.

However, it has also been shown that further criteria for gene transfer are important. This essentially includes the particle size, which may be a maximum of 200 nm, in tumor tissue for uptake from the vascular system even only 70 nm. This is on the one hand so because of the transport via vessels and tissue, on the other hand because of endocytosis in the individual cells ("Coated pits", Carter et al., 1993, Muller et al., 1995, Weigel et al., 1998, Lippincott- Schwartz, 1999), which requires strong condensation of the nucleic acid by the polycation (Godbey et al., 1999) .The interaction of the highly negatively charged nucleic acid with the positively charged polycation due to the amino groups is electrostatic the interaction is not yet fully understood, but it is likely that neutralization of the nucleic acid molecule leads to a hydrophobic collapse in which water is displaced from the complex ( 1 ).

Next the size is the Net charge of the particles important for the residence time in the system. Cationic particles, for example, are increasingly in the liver accumulated while neutral particles longer stay in the blood stream. In addition, the particles should not due activate the complement system of their charge. Already 1987 was Wu and Wu proposed polylysine-condensed DNA for transfecting cells. An important methodological advantage of polycation-nucleic acid complexes over viral ones Complex is that essential complexed longer DNA molecules can be as is the case with virus particles. This has significant implications on the type and length the genes that can be transferred.

Though show polycation-nucleic acid complexes across from Viruses have lower immunogenicity, the activation of the complement system by cationic lipids or general polycation is still noticeable. Absolom (1986) described so-called dysopsins, which prevent the proteins of the complement system the polycation-nucleic acid complexes inactivate. This includes highly hydrophilic sugar residues or polyethylene glycol (PEG), that is, covalent bound to polycation-nucleic acid complex, to a prolonged Half-life of the complex in the blood leads (Woodle et al, 1994). This is due to a "coating" of the complexes through to explain the PEG making them less vulnerable.

The previously available gene transfer systems consist of complexes of polylysine, PEI or other polycations, DNA, and a "coating" of PEG that can transfect eukaryotic cells.

Description of the invention

With The object of the present invention is to provide new combinations of Nucleic acid, Polycation, coating and specific seeker to produce such, that humane fibroblastoid cells or their precursor (stem) cells and the Receptor for specifically transfecting neuronal cells carrying the same search head become. These combinations are called "gene transfer module" (GTM) in the following.

PEI is in various molecular weight classes (MW) of about 0.7-ca. 2000 kDa commercially from different suppliers (Sigma, Aldrich, Fluka, BASF) available. According to the invention, PEI with a MG of about 2-ca. 2000 kDa used, with MG of about 2-ca. 800 kDa preferably used has been. Preferably, non-branched linear PEI became apparent more mobility is obtained in the conjugated molecules, being branched PEI is not excluded.

by virtue of The experiments for the present invention has been found that for a efficient gene transfer N: P ratios, i.e. the molar ratios of nitrogen atoms (N) of the PEI and the phosphate atoms (P) of the Nucleic acid, between 2: 1 and 48: 1 are required. The area of high efficiency is between 3: 1 and 12: 1, the most preferred range is between 5: 1 and 12: 1 for the examined primary human fibroblasts and neuronal cells, with deviations after both Directions in dependence of various parameters (cell type, degree of pegylation, seeker possible) are. In the investigated range the N: P ratios were not toxic.

to shortcut (Conjugation) of antibody and polycation, the following different strategies were tested being theoretical considerations from literature (Blair and Ghose, 1983) were.

Link under modification of the Sugar parts of the antibody:

The antibody, after oxidation with metaperiodate, is reacted with a bifunctional linker carrying an amine or, preferably, a hydrazide function (Pain and Suriola, 1981). To improve stability, the resulting azomethine is often reduced (eg with NaCNBH ₄ ). The polycation is reacted with a linker that allows the generation of the function complementary to the linker of the antibody (eg SH). It can also be coupled directly to the polycation after oxidation. Commercially, such a bridge is available, for example, from Pierce as 3- (2-pyridyldithio) propionylhydrazide (PDPH).

Link under modification of Amino groups of the antibody:

Of the antibody is using a bifunctional linker according to a protocol by Neurath and Strick (1981), in addition to an amine-reactive group (Preferably activated carboxylic acid e.g. as N-hydroxysuccinimide ester) a group for forming e.g. a thioether or disulfide bridge wearing. Further Procedure as 1. Commercially, such a bridge is e.g. available from Pierce as N-succinimidyl 3- (2-pyryldithio) propionate (SPD).

Link below Modification of the carboxylic acid function of the antibody

The carboxylic acid is activated with carbodiimide and then reacted with a corresponding linker (analogous to 1.), eg a cystamine linker [formula 1] or directly with the polycation. H ₂ N-CH ₂ -CH ₂ -SSC ₅ H ₄ N [Formula 1]

Linkage using FAB fragments

To enzymatic treatment of the antibody become FAB fragments obtained by cleavage of the C fragment with a free SH function. These are treated with a correspondingly functionalized polycation (i.e., for example, having a pyridyldithio or maleimide structure).

Preferably were in the GTM of the invention connections performed after 1st or 2nd.

The combination of polycation / nucleic acid with PEG is called pegylation in the following. As polymers which were used to pegylate the conjugates of PEI and monoclonal antibody, various polyethylene glycols were used:
Methoxy PEG Nitrophenyl Carbonate, 5000 Da Methoxy PEG Succinimide Ester, Methoxy PEG Propionic Acid, 5000 Da.

The compounds were supplied by Shearwater Polymers Inc., Huntsville, AL, USA. Commercially, such PEG esters with NH ₂ functions are available in further embodiments with different molecular weights, the molar masses being between 1000 and 6000 Da, preferably between 5000 and 6000 Da. However, this does not exclude the possibility that PEG esters with NH ₂ functions or similar, alternatively usable functions with other molecular weights can also be used.

Although it has already been shown that transfection based on polycations allows better transfection of primary human fibroblasts than with commercially available transfection agents such as Lipofectamine (Life Technologies), specific gene transfer is not possible (Zauner et al., 1999). As a search head, ie as a specific receptor for the detection of human fibroblasts monoclonal antibodies against human Thy-1 (CD90) were used. In human connective tissue, Thy-1 is only expressed on fibroblasts and precursors of these fibroblasts and - beyond the blood-brain barrier - on some neuronal cells. As the specific monoclonal antibody, antibodies which recognize this specific antigen were accordingly used. For this purpose, the monoclonal antibodies AS02 ( DE 4407242 ) or MS1 ( DE 100 06 904 ) linked via one of the above-described bridging mechanisms and used for the transfection of fibroblasts or human neuronal cells.

With the selection of AS02 or MS1 is not an exclusive restriction One of these two monoclonal antibodies intended, but can also other such specific antibodies come into question.

When nucleic acids, which are complexed with PEI, double-stranded DNA (preferably as Plasmids) into consideration. Plasmids are used when the Expression of a gene or functional gene segment in the recipient cells should be achieved.

Plasmids used are either commercially available plasmids, the signal sequences required for expression in the target cells (promoter, polyadenylation signal, selection marker, etc.) and, firstly, the amplification of the plasmid DNA in suitable bacteria (for example suitable E. coli K12 strains such as XL -1 or DH5α) ( 2 ).

Such Vectors leave a review of the Efficiency of gene transfer e.g. above the number of fluorescent cells, because a corresponding Green fluorescence gene transferred has been. In the GTM according to the invention However, preferred are those constructions in which a preferential in the target cells read promoter instead of a conventional viral, for example SV40 or cytomegalovirus, promoter functional is included. For example, the promoter for the human Thy-1 gene (Vidal et al., 1990, Szyf et al., 1990) Way target cells that are already selective about the antigen-antibody binding of the GTM were specifically transfected using a promoter, which is still largely selective for these cells. This novel Combining Thy-1 recognition at the protein and promoter levels allows one double selectivity of the GTM according to the invention.

Corresponding non-exclusive use of Thy-1-positive antibodies can, however other promoters are also used, such as promoters for human Collagen α1 (I), human collagenase 1 (MMP I) or similar connective tissue specific Promoters or so-called strong promoters, generally high Effect transcription in human cells.

When additional Splicing Element for the Transfection efficiency can optionally be a so-called "Nuclear Location Signal" (NLS), i.e. a so-called "nuclear localization signal" (NLS) short peptide, which intracellularly transports the vector relieved in the nucleus, with in the complex of PEI and nucleic acid with to get integrated. An example of such a signal is that short peptide PKKKRKV from the "Large T antigen "from SV 40, it is known that it promoted the transport of viral DNA into the nucleus selectively.

example 1

Coupling of AS02 or MS 1 to PEI via a sugar bond

The Conjugation was carried out according to the protocol of Pierce (PDP hydrazide). To 2 mg of AS02 or MS1 in 1 ml of phosphate-buffered physiological Saline (PBS) is added 0.1 ml of cold sodium periodate solution and 20 minutes at 0 ° C incubated in the dark. Subsequently glycerol is added to a concentration of 15 mmol / l and 5 minutes at 0 ° C incubated to stop the oxidation. After that 16 hours against 0.1 mol / l sodium acetate buffer, pH 5.5, dialyzed. PDP hydrazide solution (20 mmol / l in DMSO) to a final concentration of 5 mmol / l and Incubated for 2 hours at room temperature with shaking. Unconjugated Linker was over a desalting column away. Similarly, PEI MG 25000 was conjugated to PDP hydrazide. The PDP hydrazide activated PEI was then treated with dithiothreitol (DTT) reduced by adding 12 mg DTT in 500 μl acetate buffer (100 mmol / l sodium acetate, 100 mmol / l NaCl, pH 4.5) to 1 ml of the PDP hydrazide protein and 30 min. was incubated. It was then desalted on a desalting column, fractionated collected and the amine group concentration determined. The SPDP-activated antibody was afterwards for Stirred for 18 hours at room temperature with the DTT-coupled PEI. The conjugate was then subjected to size exclusion chromatography fractionated and over Microcentrifugation filter (Centricon-30, Amicon) concentrated.

Example 2

Coupling of AS02 or MS 1 to PEI via SPDP

The Conjugation was performed according to the protocol of Pierce (SPDP). To 10 mg of AS02 or MS1 in 1 ml of phosphate-buffered physiological Saline (PBS) were 25 μl SPDP solution (20 mmol / L) and 30-60 Incubated for a few minutes at room temperature. Unconjugated linker was over one desalting column away. Accordingly, PEI MG 25000 was conjugated to SPDP. The SPDP-activated PEI was subsequently reduced with dithiothreitol (DTT) by adding 12 mg DTT in 500 μl acetate buffer (100 mM sodium acetate, 100 mmol / L NaCl, pH 4.5) to 1 ml of the SPDP protein given and 30 min. was incubated. It was then desalted on a desalting column, fractionated collected and the amine group concentration determined. The SPDP-activated antibody was afterwards for Mixed for 18 hours at room temperature with the DTT-coupled PEI. The conjugate was then subjected to size exclusion chromatography fractionated and over Concentrated microcentrifugation filter.

Example 3

Complexation of the PEI / antibody complex with nucleic acid

The complexes are prepared by plasmid DNA, for example, the in 2 represented plasmid pFACSort (Peterssohn, D., Dissertation 1996, University of Cologne), which is a review of the transfection efficiency without further introduced genes except a GFP (green fluorescence) under the control of an EF1α- Promoter allowed to be mixed with conjugated PEI (see Example 1 or 2). 3 μg of plasmid DNA are mixed with the conjugated PEI / antibody complex depending on the N: P ratio of 8: 1-10: 1 and mixed with 20 mmol / l HEPES pH 7.3 / 150 mmol / l NaCl to a total volume of 115 μl. By 15-25maliges up and pipetting the complex formation is promoted. Subsequently, the complexes form during a 20 minute incubation at room temperature. The mixture of PEI / antibody / DNA is now either used directly for transfection or previously pegylated. For this purpose, either 5-7.5 μg of methoxy-PEG-nitrophenyl carbonate, 5000 Da or methoxy-PEG-succinimide-propionic acid-PEG, 5000 Da, in each case 10 mg / ml in DMSO, in 50 μl of ddH ₂ O are added to the mixture and incubated for 20-30 minutes at room temperature. The ratio of PEG to PEI (w / w) is in the range of 6-10). Attempts have also been made to pegylate the PEI / antibody complexes prior to addition of the nucleic acid, which also functioned in principle but, presumably due to greater particle size variation, resulted in lower transfection efficiency.

Example 4

Transfection of human fibroblasts

Transfection complexes according to Example 3 were brought to a final volume of between 500 and 600 μl with cell culture medium, for example RPMI or DMEM with 2% fetal calf serum, and to the human fibroblasts to be transfected the day before in a number of 10000 to 35000 cells / well Well culture plate were pipetted evenly after removal of the culture medium present on the cells. The transfection complexes were rapidly brought into close contact with the cells by centrifugation (5 minutes, room temperature, 276 x g). After incubation for 1.5 to 4 hours (37 ° C, 5% CO ₂ ), the transfection mixture was removed, the cells rinsed with 1 ml of medium without additives and further incubated with medium containing 10% fetal calf serum and penicillin / streptomycin according to standard protocols. After a maximum of 20 hours of incubation, transfectants are observed in the fluorescence microscope. Compared to a commercially available transfection agent (TransFast, Promega, Mannheim), at least three times higher transfection efficiency could be observed.

Example 5

Transfection of mixed human cells

One to Example 4 analogous approach was for the transfection of a 1: 1 mixture of human endothelial cells and primary human fibroblasts.

normal human endothelial cells do not express Thy-1. It could be observed be that the Number of transfected fibroblasts that of the transfected Endothelial cells regularly around many times exceeded, at least by a factor of 50 to a factor of 200. Corresponding control experiments with PEI / nucleic acid / PEG with an irrelevant nonspecific antibody showed no such Differences.

Example 6

Transfection of human neuronal cells

An approach analogous to Example 4 was carried out for the transfection of human neuroectodermal sarcoma cells. Also in the case of neuronal cells, a significantly increased transfection rate could be measured in transfection batches containing the combination PEI / AS02 / DNA / PEG the.

references

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Claims (13)

A method for directed gene transfer into human fibroblastoid cells, characterized in that polyethyleneimine having a molecular weight <2,000 kDa is reacted with a plasmid containing the tissue-specific promoter Thy-1 or a nuclear localization signal, such that the ratio of the nitrogen of the polyethyleneimine to phosphorus the nucleic acid is 2 to 1 to 48 to 1, the resulting construct is reacted with a monoclonal antibody to recognize human fibroblasts such that the linkage is carried out with modification of the sugar moiety of the monoclonal antibody or its amino group, the conjugate is pegylated and used for gene transfer , Method according to claim 1, characterized in that that as a monoclonal antibody the AS02, produced by the hybridoma DSZM ACC 2164 or the monoclonal antibody MS1, produced by the hybridoma DSZM ACC 2402, or other, human Thy-1 recognizing monoclonal antibodies are used. Process according to claims 1 and 2, characterized that the plasmid is a double-stranded DNA plasmid is. Method according to claim 1, characterized in that that linear PEI is used. Method according to claim 1, characterized in that that the ratio of the nitrogen of the PEI to the phosphorus of the nucleic acid of 5 to 1 to 12 to 1 is preferred. Method according to claim 1, characterized in that that for linking a functional linker, in particular 3- (2-pyridildithio) propionylhydrazide or N-succinimidyl-3- (2-pyridildithio) propionate, as a bridge is used. Gene transfer module consisting of a plasmid, which is the tissue-specific promoter Thy-1 or a nuclear localization signal contains polyethyleneimine with a molecular weight <2,000 kDa as a polycation, a bifunctional linker as a bridge, one monoclonal antibody for recognizing human fibroblasts, wherein the conjugate is polyethyleneimine, bridge and monoclonal antibody is reacted with polyethylene glycol. Gene transfer module according to claim 8, characterized in that that serves as a polycation linear polyethyleneimine having a molecular weight <800 kDa. Gene transfer module according to claims 7 and 8, characterized that the bridge is a difunctional amine or hydrazide. Gene transfer module according to claims 7 and 9, characterized that the bifunctional linker next to an amine-reactive group carries a group to form a thioether or disulfide bridge. Gene transfer module according to claim 7, characterized that as a monoclonal antibody the AS02 from the hybridoma DSZM ACC 2164 or MS1 from the hybridoma DSZM ACC 2402 serves. Gene transfer module according to claim 7, characterized that the nuclear Localization signal is a short peptide, which intracellularly transport the vector is supported in the nucleus. Gent transfer module according to claim 12, characterized in that that the peptide contains between 5 and 10 amino acids.