DD256148A1 - PACKAGING METHOD OF DNA FOR GENTRANSFER TO ANIMAL CELLS - Google Patents

Abstract

The process according to the invention for packaging DNA for eukaryotic gene transfer triggers conventional methods of transfecting cells with foreign genes. By transferring the sequence-specific sequence of the vector into an active form with chromosomal proteins and taking it up in monodisperse form from the recipient cells, high transfection rates result. An additional condensation of the vector leads to a further improvement of the results. The method is also suitable for combination with other transfection methods.

Description

Field of application of the invention

The invention relates to a method for packaging DNA for eukaryotic gene transfer. The field of application is molecular biology and biomedical research as well as biotechnology-based drug production.

Characteristic of the known technical solutions

Various methods exist for transferring genetic material into cells in vitro and expressing them there. The most important are calcium phosphate DNA coprecipitation and transfection with the aid of polycations, which are characterized by easy handling. However, as this method has a number of disadvantages, new methods are constantly being published.

The use of these methods is often adapted to specific objectives and often requires a considerable amount of experimental and equipment. Little is known about the mechanisms leading to DNA uptake, transport through the cytoplasm, and expression in the nucleus. A comprehensive review containing a detailed bibliography has recently been published (M. Strauss, U. Kiessling, M. Platzer [1986] Biol. Zentralbl. 105, 209-243). The method of calcium phosphate-DNA coprecipitation is most commonly used because of its ease of handling and good reproducibility. The DNA to be transfected, which is in a CaCl ₂ solution, is added to a phosphate buffer solution, with DNA and calcium phosphate forming common precipitates. The precipitates sediment after addition to the culture medium on the cells, where they are absorbed and absorbed by phagocytosis. The essential prerequisite is that close optimal conditions with respect to the pH, the DNA concentration and the size of the precipitates are met. By using carrier DNA and applying a glycerol or DMSO shock, the yield of transformed cells may be improved. The following disadvantages of the method must be considered:

1) Only a small cell fraction is stably transformed under optimal conditions.

2) The foreign DNA is taken up unphysiologically as a precipitate by phagocytosis and is probably transported to the nucleus in lysosomes or other cellular compartments. The acidic environment of the lysosbms could lead to depurinization of the DNA and thus to deletion mutants. In addition, most of the DNA in the cytoplasm is degraded.

3) In the cell nucleus, only part of the DNA is packed in chromatin, which is a prerequisite for their activation and protection against nuclease.

4) Dietary transfected DNA resides in the nucleus as a transgenom of hundreds of copies of DNA into the host core DNA, suggesting an unphysiological integration mechanism. The extent of gene expression is generally not increased thereby.

One variant of the calcium phosphate-DNA coprecipitation method is transfection with the aid of polycations, of which DEAE-dextran has found the widest application. Electrostatically-induced complex formation, based on the polyanionic nature of DNA, facilitates its absorption at the cell surface. The uptake by the cells is likewise by phagocytosis. The complexes also provide some nuclease protection. The success of the method depends critically on the concentration of the polycation and its residence time on the cells. The method is particularly useful for short-term expression assays where transient gene expression from non-replicating vectors is investigated and exploited. Most of the disadvantages of coprecipitation also apply to this method.

Similar efficiency to the coprecipitation method is obtained after inclusion of DNA to be transfected into liposomes. The DNA is introduced into the cell interior after fusion of the lipid membrane of the liposomes with the cell membrane. By incorporation of antibodies or glycolipids into the lipid membrane, selectivity for certain cell types can be achieved with this method. Here and in the possible application to the in vivo gene transfer z. B. by injection into the blood or lymphatic system, the benefits of this method can be seen. A disadvantage is the complicated preparation of the liposomes.

The method of electroporation is based on the fact that electric fields increase the membrane permeation of the DNA by inducing transient membrane pores. Damage caused by the cells and in particular the membranes can not be excluded. The method is particularly suitable for use in poorly cultured cells, such as lymphocytes or hematopoeitic stem cells. Prerequisite is an apparatus that generates short-term electrical "field jumps".

The most effective DNA transfer method is the microinjection technique in single cells. The DNA is injected directly into the nucleus by microcapillaries with an outer diameter of 0.5μιη, which requires a considerable amount of equipment and great manual skill. The method is used when high-efficiency foreign DNA in single cells, eg. B. unicellular embryos is to be introduced and therefore can not be compared with the other methods shown here. A critical evaluation of the o. A. Methods are also described in the aforementioned review by Strauss et al. and in another review by H. Hauser (1985) in Molecular and Cell Biology, Ed. A. Blin et al., Springer Verlag, Berlin, pp. 159-176. Thus, the available methods prove more or less well adapted to particular cell systems or experimental arrangements. However, the individual methods are not universally applicable. This could be attributed to the fact that the foreign DNA is taken up in most cases as an unphysiological precipitate or aggregate or in complex with unphysiological substances from the cell or the cells must be physically manipulated. Significant improvements in both the efficiency and scope of various cell systems are expected in packaging the DNA, which is closer to cellular conditions.

Object of the invention

The aim of the invention is to develop by the introduction of physiological transfer conditions an effective transfection method, which is convenient to combine with other methods and thus opens up new applications. The method should be easy to handle and also suitable for industrially relevant recipient cell lines.

Presentation of the essence of the invention

The invention has for its object to introduce by a suitable DNA packaging method foreign genetic information in the form of plasmid or viral vectors in recipient cells and bring in Zellkerfi for expression. According to the invention, the object is achieved by subjecting a vector to a sequence-specific liganding with specific chromosomal proteins and thereby converting them into a state suitable for gene activation. By transferring the vector into a condensed form, in addition the uptake by the cells is facilitated and an effective nuclease protection is achieved

 The method according to the invention is characterized by the following features:

a) The expression control or regulatory regions of a vector are added to a suitable state for gene activation prior to ingestion by the recipient cells by liganding. The applied liganding protects these regions against histone packaging in the cell nucleus or the formation of a modified histone packaging accessible for recognition processes, whereby the genes to be transferred are already functionally actively introduced into any host cells.

b) By applying the same proteins in excess or other chromosomal proteins and / or optionally divalent cations, the vector is additionally condensed and stabilized, the condensed complexes, in contrast to other methods, being present in monodisperse form with respect to the DNA.

The complex formation of the vector DNA with the proteins, which leads to the liganding and condensation of the DNA, is carried out by slow stepwise mixing or by dialysis. This avoids the formation of insoluble aggregates, which leads to DNA losses. Decondensation of the complexes by dilution, as occurs in the treatment of the cells, is prevented by the use of the proteins in excess or by the presence of divalent cations, preferably CaCl ₂ .

In this way, the transfection process is facilitated by physiological conditions without toxic substances having to be introduced for the cells. The condensation or packaging of the DNA in monodisperse form also allows the combination with other transfer methods, such as inclusion in liposomes, electroporation and microinjection.

According to the invention, the HMG proteins (high-mobility-group) 1 and 2 and the nucleosomal histones H2A, H2B, H3 and H4, which are used, for example, as suitable chromosomal proteins. from calf thymus or rat liver by known methods (E.W. Johns, G.H. Goodwin, J.M. Walker, C. Sanders (1975) Ciba Found, Symp., 28.95-100). Also suitable are equivalent HMG proteins from other organisms (eg HMG E, HMG T) or generally chromosomal proteins with similar properties. HMG1 and 2 are very similar proteins with a molecular weight of about 25,000,

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which have a pronounced domain structure (GR Reeck, PJ Isackson, DC Teller (1982) Nature 300, 76-78). Two folded basic domains interact with the DNA in preferential electrostatic interaction, a strongly acidic domain at the C-terminus mediates a good solubility of the complexes, an important property in this context (M. Carballo, P. Puigdomenech, J.Palau [1983] EMBO -J.2 , 1759-1764). HMG1 and 2 preferentially bind to single stranded DNA but also show high affinity for superhelical DNA (C. Bonne, M. Duguet, A.M. de Recondo [1980] Nucleic Acids Res. 8, 4955-4968). In the DNA complexes, both HMG proteins bind to regions of altered DNA secondary structure under appropriate conditions. In addition to single-stranded regions BZ compounds and Kruziformstrukturen come in. Question (H.Hamada, M. Bustin [1985] Biochem 24.1428-1433). These structures are of interest as a possible component of the control region of genes. In addition to binding to free DNA, HMG1 and 2 may also be part of active nucleosomes, ie, transcribed chromatin (JB Jackson, RL Rill [1981] Biochem., 20, 1042-1046). The biological mode of action for steps a) and b) is explained by the following experiments:

The complex formation between HMG1 and the vector pLTEneo (6.8 kBP), which is given as an example here, is analyzed in an analytical ultracentrifuge Spinco E (Beckman) with the aid of UV absorption optics. A detailed molecular genetic description of the vector is not required. It should be noted that it is under the control of 3 Ori regions (Py, SV 40, pBR322) and carries the neo gene for selection purposes. The complexes are prepared by stepwise addition of the protein to the centrifuge cell containing and interacting with the DNA and characterized for its sedimentation behavior and relative protein binding. The latter is expressed as the extinction ratio E23o / E26s of the sedimenting, i. expressed in bound material. The buffer used is 0.15 M NaCl, 10 mM Tris-HCl, pH 8. The results are shown in Fig. 1. It can be seen that the sedimentation coefficients of both DNA ring components increase with increasing HMG1 addition ratio η and coincide at about η = 10. At even higher η values, no significant s value changes are observed. Precipitation was not detected.

• *

(GHMG1 / gona)

12

Fig. 1 Sedimentation and relative binding measurements of the superhelical I as a function of the HMG 1 addition

\ And open (II) ring shape of the vector pLTEneo

From this it can be concluded that the structure of both DNA forms is the same after complex formation at r- > 10. The binding measurements, on the other hand, led to the conclusion that saturation is already reached at η> 4, ie that the s-value increase in> 4 is largely due to condensation of the complexes and not to protein binding. The proteins are here for the most part unbound in excess.

Figure 2 shows the stability of these complexes of 70-80S versus dilution with various solvents. Strong dilution generally results in the dissociation of electrostatic interaction-based complexes. The complex starting concentration based on DNA was 20-40 μg / ml.

It can be seen that dilution with water or buffer leads to dissociation and / or decondensation of the complexes, whereby the starting s values of the free DNA are not yet reached. By contrast, dilution with an HMG1 solution of 0.5 mg / ml in the above buffer or a CaCl ₂ solution up to a final concentration of 12.0 mM CaCl ₂ resulted in stabilization of the complexes.

100 I i ·· - · I ι i '' I X I - I 8Oi ::: ".:.; ^r " CaCf ₂ __ ^ ' a.C3 "ω" 60 "'"' "Cx Na Cl ' Sparmin \ -._ \ a - .. 0.1SM - 40 • m on I ι I ι I I

0.8

0.6

Dilution

0.4

0.2

Fig.2 Stability of complexes of the vector pLTEneo with HMG1 versus dilution with different media

From these experiments it can be concluded that an excess of unbound HMG1 or the presence of soluble calcium stabilizes the compact structure of the complexes upon dilution.

The complexes were diluted after dilution (1: 10-20) with the o.a. Püffer, d. H. in the decondensed state and / or after dissociation unspecifically or loosely bound protein by the Dubochet method electron microscopy shown (Philips EM 400T). A fixation of the material with glutaraldehyde proved necessary. Proteins bound to 1-3 sites of DNA were detected in the form of nucleosome-like particles. In order to obtain information about a possible sequence specificity of this binding, these complexes were once specifically cleaved with the restriction endonuclease Bam H1 and the distances of the bound proteins from the cleavage site, which proved to be reproducible and thus sequence-specific, measured. The comparison with a functional map of the vector is shown in Fig. 3. It can be seen that HMG1 binds to 3 regions of the vector pLTEneo, which i.a. are characterized by the presence of the 3 Ori regions and the early polyoma genes, within these regions HMG1 binds particularly significantly to the Oris itself, as indicated by the narrow abundance maxima. This demonstrates selective binding of HMG1 to control and regulatory regions of genes at physiological salt conditions. From Fig. 3 it can also be seen that there is no AT specificity of the binding. This electron microscopic method is also suitable for finding other suitable selective binding proteins. *

6.8 kb

SVW

pBR322

Py

6,8kb

EcoRI

Fig.3 Frequency of HMG 1 binding to selective sections of the pLTEneo genome, comparison with AT sequences and comparison with a simplified functional map of the vector

Transfection experiments with such complexes, which are shown here as application examples, showed the result that already in the decondensed state of the complexes after dilution with buffer a similar or better transfection efficiency than with the conventional Kopräzipitationsmethode can be achieved (see Table 1). Thus, the specific liganding of the control and regulatory sequences of vectors is of particular importance in transfection. Transfection efficiency can be further increased when the complexes are in condensed form, i. H. in excess of HMG1 or in the presence of soluble calcium from the host cells. Herein expresses the improved membrane permeation of condensed complexes and effective nuclease protection (K. Shastri, P.J. Isackson, J.LFishback, M.Daniel, G. Reeck [1982] Nucleic Adids Res. 10, 5059-5072). Accordingly, HMG1 protects in vitro against single strand-specific nucleases and DNase I.

It was thus shown that in the transfection system introduced here, the control regions which are relevant for the function of the vectors are functionally liganded and converted into a state which can be addressed for the downstream regulatory processes. The introduction of the foreign gene vector ligand system takes place without mechanical or electrical cell damage in monodisperse form and is thus independent of the recipient cell type. This meets the above stated objective

 Subsequently, the invention will be explained in more detail by way of examples.

embodiments

1. HMG 1-induced transfection of mouse I cells with the vector pLTEneo vector and HMG1 are mixed at physiological salt concentration and room temperature so that after repeated addition of HMG1 in portions to the DNA HMG1 / DNA ratio π in g HMGVg DNA of about 10 is reached. After each addition of HMG 1, slowly swirl the sample and leave it at room temperature for about 40 minutes. The mixture must be made slowly or stepwise in small portions to avoid precipitation of the complexes. The addition of larger amounts of HMG1 may result in turbidity of the solution and hence in DNA losses due to precipitation. Incubation of

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Mixture at 37 ^° C. gives no improvement. Dialysis of the mixture of both components, which are then initially dissolved in 2 M NaCl, can also be carried out against saline solutions of decreasing concentration and finally against the abovementioned buffer. However, because of the good solubility of the complexes, this procedure is not necessary, usually 0.2 ml pLTEneo a concentration of 60-80jug / ml, dissolved in 0.15M NaCl, 1OmM Tris-HCl, pH 8, with 5 portions HMG1 each 50 μ \ a concentration of 0.5 mg / ml mixed in the above buffer. The material should then sediment as a uniform component with about 80S, which can be determined by a sedimentation velocity test in an analytical ultracentrifuge (see Fig. 1). The mixture can also be carried out in an ultracentrifuge cell in order to be able to control the course of the complex formation immediately.

For the purpose of transfection, the complex can be applied to the cells by various methods. In order to achieve high transfection rates, the complex should be stabilized in the condensed state despite the dilution required for addition to the cells (see Fig. 2). However, satisfactory transfection rates are also obtained in the decondensed state. The following variants are possible:

1.1. 0.25 ml of complex solution with a sedimentation coefficient of 70-80 S and a DNA content of 6 ^ g are diluted 1:10 with culture medium and added to about 10 ^B mouse L cells. In this case, a recording of the complex by the cells in decondensed form is to be expected.

1.2. The same amount of 75 S complex is diluted 1:10 with culture medium to which 0.5 ml HMG 1 solution at a concentration of 2.5 mg / ml has been added. According to Fig. 2, the condensates are retained in this form of application.

1.3. CaCI ₂ is added to the culture medium in a final concentration of 12, OmM. The dilution of the 75S complex is carried out as in a) and b). Again, the condensates are largely retained.

The results of a representative transfection experiment are summarized in Table 1. As a control, the calcium phosphate-DNA co-precipitation method was used in the absence of HMG1. It can be seen that in the presence of HMG1, but under non-condensing conditions (dilution of the complex with culture medium), an increased transfection rate compared to the calcium phosphate method is achieved (1.1.). At an even higher free HMG1 concentration, i. H. under condensing conditions, it continues to rise (1.2.). Free DNA in the absence of HMG1 or calcium is not absorbed by these cells.

Table 1: Transfection of mouse L cells with pLTE neo

Calcium phosphate colonies transfection rate ⁰¹

pLTEneo ³¹ + 180 1.8 · 10 ~ ³

Control - 0 0

HMGI complex ³¹ · " ¹

+ Culture medium * - about 300 about 3-10 ~ ³

HMGI complex ³ '× " ¹

+ Culture medium with HMG 1

(Final concentration 0.05 mg / ml) - S 500 approx. 5-1O ^-3

a) 6μg DNA, b) 75S complex diluted 1:10; HMG1 / DNA (w / w) = 10: 1 c) number of colonies / 10 ⁵ cells

2. Transfection of mouse L cells with the vector pCGBPV Z10 using HMG1 and the histones H 2A, H 2 B, H 3 and H 4 To determine if for the observed HMG 1-induced transfection the condensation of the vector or the specific binding to the expression control regions of the vector is also responsible, the transfection efficiency of histone-DNA complexes in the form of reconstituted minichromosomes and complexes of HMG1, 4 nuclear histones H 2A, H2B, H3 and H4 and the vector pCGBPVZIO was also examined. Histone-DNA complexes in the form of minichromosomes represent a natural condensation form of DNA. The reconstituted minichromosomes, which have a high degree of condensation, proved to be transfection-inactive in the absence of kaziumphosphate (S. Scherneck, H. Selected, M. Theile, M. Böttger, CU of Mickwitz, E. Geissler [1983] Acta virol., 27: 1-11). The 4 core histones were used as an equimolar mixture and applied to the DNA in a ratio of 1.5: 1. A high DNA condensation alone, even in the form of chromatin-like complexes, is therefore not sufficient to transform cells.

In contrast, HMG1 histone-DNA complexes are transfection-active. Such complexes were prepared following previously published conditions under which the HMG1 protein supports the assembly of nucleosomes (Bonne-Andrea, F. Harper, J. Sobczak, A.-M. de Recondo [1984] EMBO-J .3,1193-1199). The complexes are prepared by mixing the constituents in 0.15M NaCl, 10 mM Tris-HCl, pH 8 in the weight ratio HMG 1 / histone / DNA = 3: 1.5: 1, the histones being used as an equimolar mixture. First, stock solutions of histones and HMG1 are mixed, with a slight turbidity occurring, which is removed by low-speed centrifugation at 3000 rpm for 15 minutes. An incubation of the mixture at 37 ⁰ C brings no improvement. Subsequently, the DNA is added very slowly with gentle swirling. Again, a turbidity is often observed, which can also be removed by low-speed centrifugation. The entire procedure can be carried out in the measuring cell of an analytical ultracentrifuge, whereby the formation of aggregates and their removal at wavelengths of 230 nm (protein) and 265 nm (DNA or complex) is monitored by means of the absorption optics.

The formation of complexes can be proven by their sedimentation behavior. Thus, for pCGBPV Z10, an increase in sedimentation coefficients s ₂ JS? From 27.6S for Form I DNA to 75-99S and from 20.4S for Form II DNA to 61-76S was measured, depending on the experimental approach. The fluctuation range of the s-values of the complexes can be traced back to changing amounts of precipitated protein.

Table 2: Transfection of mouse L cells with pCGBPVZIO pCGBPVZ10 ^a) Reconstituted

Minichromosomes a, b)

+ Ca -Ca * + Ca -Ca

^100cl 0 125 120

b) HMG 1 / histone / DNA (w / w / w) = 3: 1.5: 1,

c) Number of colonies / 10 ⁵ cells

The results of a TranSfektionsversuchs with these complexes are shown in Tab. 2. It can be seen that in the presence of HMG1 a similar yield of transformed colonies is achieved as after application of the calcium phosphate co-precipitation method to the protein-free vector or complex. Thus, it can be concluded that the non-histone protein HMG1 is responsible for the transfection activity of the complexes described herein and that the principle of the sequence-specific liganding of the control regions of the vector according to the invention is of crucial importance.

Claims (5)

1. Packaging DNA for gene transfer into animal cells, characterized in that a vector is ligated by means of functional-chromosomal proteins in a sequence suitable for gene activation, the vector by interaction with these proteins in excess or by other chromosomal proteins and / or or optionally converted divalent cations in a transfection-active condensed complex, stabilized against decondensation by dilution and introduced in monodispersed form in any host cells. 2. The method according to item 1, characterized in that the sequence-specific liganding and the condensation is carried out by slowly stepwise mixing of the vector DNA and the proteins without turbidity. 3. The method according to item 1, characterized in that the sequence-specific liganding and condensation takes place by dialysis. 4. The method according to item 1-3, characterized in that the non-histones HMG1 and HMG 2 and mixtures of these proteins with the histones H 2 A, H 2 B, and H 4 are used as functional chromosomal proteins. 5. The method according to item 1-4, characterized in that the optimal transfection concentration of the vector is prepared by dilution with culture medium to which HMG1 or CaCl _{2 is} added.