WO2004020615A1

WO2004020615A1 - Method for the production of virus-analog particles for packing biologically active macromolecule by in vitro assembly of capsomers to which the macromolecules are bound

Info

Publication number: WO2004020615A1
Application number: PCT/DE2003/002688
Authority: WO
Inventors: Rainer Rudolph; Hauke Lilie; Thomas Jahn; Stefan Gleiter; Kay Stubenrauch
Original assignee: Martin-Luther-Universität Halle-Wittenberg
Priority date: 2002-08-13
Filing date: 2003-08-09
Publication date: 2004-03-11
Also published as: DE10237639A1; DE10393561D2; AU2003263132A1; EP1539939A1

Abstract

Virus-analog particles are predominantly produced recombinantly. Recombinant expression of capsomers in Escherichia coli makes it possible to obtain non-assembled capsomers which can be assembled in vitro. However, the degree of assembly is low. In fact, in vitro assembly into virus-analog particles is prevented if the macromolecules are bound to the capsomers. Complexes with macromolecules can only be obtained by interaction of the macromolecules with the already assembled virus-analog capsomers. The degree of packing of the macromolecules is nevertheless low. Virus-analog particles are produced by means of a method for the production of virus-analog particles by in vitro assembly of capsomers or capsomers, biologically active macromolecules being bound to the charged amino acids of said capsomers with simultaneous packing to macromolecules in the presence of non-ionic stabilizers. Said complexes are suitable vectors for gene therapy.

Description

METHOD FOR PRODUCING VIRUS ANALOG PARTICLES FOR PACKING BIOLOGICALLY ACTIVE MACROMOLECULES BY IN VITRO ASSEMBLY OF CAPSOMERS TO WHICH THE MACROMOLECULES ARE TIED

The invention relates to a method for the in vitro assembly of capsomers or of capsomers, to which biologically active macromolecules are bound, into virus-analogous particles or for packaging biologically active macromolecules into these virus-analogous particles during in vitro assembly. These complexes represent vectors suitable for gene therapy. They make it possible to transduce biologically active macromolecules in cells so that they are functional there. In this case, capsomeres refer to naturally occurring virus envelope proteins, recombinantly produced virus envelope proteins or genetically engineered variants of virus envelope proteins.

Virus-analogous particles, hereinafter abbreviated as VLP's, represent a virus envelope consisting of one or more virus envelope proteins. They can take various forms; in the case of the VLPs derived from Polyoma VP1, for example hollow spheres with a diameter of approximately 40-50 nm, which are composed of 360 VP1 molecules arranged in 72 pentamers. In addition to this type of VLP's, Polyoma VP1 can also form smaller shapes and rod-shaped VLP's (Salunke et al., 1989, Biophys. J. 56, 887-900). The VLP's can be produced by the recombinant production of the capsomeres in insect cells or yeast. VLPs are isolated directly in this production. The recombinant expression of capsomeres in Escherichia coli leads to the recovery of non-assembled capsomeres, which can be assembled in vitro to VLP's. This assembly process is often carried out in the presence of a stabilizing salt such as ammonium sulfate. Thus, Polyoma VP1 is assembled under these conditions to form homogeneously structured VLPs. The use of required high concentrations of this salt in the assembly results in high ionic strengths of the buffer, which is binding of nucleic acids and other biologically active macromolecules affecting the capsomeres. Ammonium sulfate is therefore unsuitable for use in the packaging of biologically active macromolecules associated with the assembly of capsomers to which macromolecules are bound.

For example, the capsomer of polyomavirus VP1 is able to bind biologically active macromolecules. A binding site of the protein for nucleic acids is located in the N-terminal sequence, which contains a high proportion of positively charged amino acids (Moreland et al., 1991, J. Virol. 65, 1168-1176). Other macromolecules can also bind to this sequence. The binding of Polyoma VP1 to plasmid DNA prevents in vitro assembly into VLP's. Therefore, the current procedures of. Complex formation of Polyoma VP1 with plasmid DNA on the interaction of already assembled VLP's with DNA. The procedure described leads to an interaction of VP1 and DNA, in which the DNA is partially protected against DNase digestion. With these complexes cells can be transduced both in vitro and in vivo. However, the efficiency of the complex formation of VLPs and DNA and the degree of DNase protection have proven to be very low (Forstova et al., 1995, Hum. Gene Ther. 6, 297-306). This is due to the fact that plasmid DNA is only incorporated into the VLPs to a very small extent when incubated with VLPs, the majority of the DNA bound by VLPs remains outside the VLPs and is at best associated with the surface of the VLPs without being packaged , Comparable results were also obtained for the packaging of oligonucleotides. In both cases, the efficiency of the complex formation was significantly improved by lowering the pH during the complex formation to pH 5, but this method does not lead to packaging of the nucleic acids used. In order to specifically cover proteins in VLP's, a WW domain was fused to the N-terminus of Polyoma VP1, which can bind proline-rich sequences. With this variant, GFP, to which a proline-rich sequence was fused, could be bound and packed into the VLPs (Schmidt et al., 2001, FASEB J. 15, 1646-1648).

The invention is based on the problem of developing a new method for the in vitro assembly of capsomers in VLPs or for packaging biologically active macromolecules during the in vitro assembly of capsomers, in which a high efficiency of the assembly and a high degree of packaging for biologically active Macromolecules is achieved.

The problem is solved in that capsomers or capsomers, to which biologically active macromolecules are bound by means of ionic interactions, are assembled in the presence of an oxidizing redox system and non-ionic stabilizers in a buffer with an ionic strength below 250 mM. Suitable macromolecules are, in particular, but not exclusively, nucleic acids such as double-stranded DNA, double-stranded RNA, single-stranded DNA and single-stranded RNA, PNA's, proteins or peptides. The size of the nucleic acid to be packaged is preferred, but not exclusively 10 to 5400 bases, preferably 20 to 1000 bases.

Viral envelope proteins that can be assembled in vitro to VLP's come into consideration as capsomers. This is described for virus envelope proteins or capsid proteins from, for example, polyoma and related viruses, papilloma virus, poliovirus, hepatitis virus, lentiviruses, Rous Sarcoma virus or adeno-associated virus. The process is based on the surprising effect that the non-ionic stabilizers

Favor assembly of capsomeres to VLP's. As a non-ionic stabilizing

Substances can serve sugar with C3, C4, C5 and C6 units such as glycerin,

Trehalose, fructose or sorbitol, but also disaccharides such as sucrose or galactose and

Oligosaccharides such as amylose or amylopectin.

As an alternative to sugars, polyols such as ethylene glycol or

Polyethylene glycol of various chain lengths can be used.

The non-ionic substance is added to the buffer in concentrations of 5-50% (w / v), preferably in the concentration range of 15-30% (w / v).

Capsomeres or capsomeres with bound macromolecules are assembled in a concentration range from 50 μg / ml - 5 mg / ml to VLPs, preferably from 0.25 - 2 mg / ml, at a pH of pH 7-8.5 and temperatures between 15 and 30 ° C. preferably pH 7.2 - 7.5 and a temperature of 20 - 25 ° C. Tris, Hepes and phosphate, for example, can be used as buffer substances, preferably in concentrations of 10-100 mM.

The assembly is carried out under oxidizing conditions. This can include the oxidation by molecular oxygen or, preferably, the oxidation using a redox system composed of oxidized and reduced thiol-containing substances such as reduced and oxidized glutathione, cysteine / cystine, cysteamine / cystamine, reduced / oxidized β-mercaptoethanol or aromatic thiol-containing substances. These are preferably used as a mixture of oxidized to reduced substance in a ratio of 1:10 to 10: 1 or by exclusive addition of the oxidizing substance.

In the method according to the invention for assembling capsomeres, the capsomeres to be assembled or the capsomeres to which contact is to be brought into contact biologically active macromolecules are bound to the assembly buffer by diluting the capsomers with the assembly buffer and / or dialyzing. Basically, a rebuffering of the capsomeres into the assembly buffer must be guaranteed.

The packaging of biologically active macromolecules during assembly is preferably carried out by incubating the macromolecules with the capsomeres. The protein concentration is in the range of 0.05 - 5 mg / ml, preferably 0.25 - 2 mg / ml.

The macromolecules bound to the capsomeres are packaged with high efficiency during assembly into the resulting VLPs. This concerns both the amount of packaged macromolecules per VLP and the relative proportion of VLP's in which macromolecules were packed. To check the packaging of biologically active macromolecules in VLP's, the unpackaged macromolecules are digested with the help of DNases, RNases or proteases and the packaged and thus protected from degradation is quantified using agarose gel electrophoresis or chromatography. It is particularly important to ensure that macromolecules that are non-specifically associated with the VLP's are also broken down. This can be analyzed by determining the efficiency of the packaging as a function of the DNase, RNase or protease concentration used. The present invention is described in the following with reference to examples. The following figures are referred to:

Illustration 1 :

Assembly of Polyoma VPl to virus-analogous particles in vitro

Figure 2:

Packaging of DNA by in vitro assembly of VPl

Figure 3:

Binding of double-stranded DNA to VPl

Figure 4:

Quantification of dsDNA binding to VPl

Figure 5:

Influence of ionic strength on the binding of dsDNA

Figure 6:

Binding of single-stranded DNA to VPl

Figure 7:

Binding of single-stranded RNA to VPl Example 1: Assembly of Polyoma VP 1 to VLP 's in vitro

Polyoma VPl was dialyzed at 20 ° C for 2 days against 20 mM Tris, pH 7.4, 1 mM CaCl ₂ ,

25% sorbitol (w / v), 0.5mM GSH, 5mM GSSG. The protein concentration was 0.5 mg / ml.

The formation of the VLPs was then checked with the help of analytical

Ultracentrifugation and electron microscopy. For analytical ultracentrifugation, the sample was centrifuged at 20 ° C. and 20,000 rpm in an AnTi50 rotor. The distribution of the VLPs in the measuring cell was measured every 10 minutes by scanning the sample at 280 nm. The sedimentation of the protein determined from this shows that VPl is assembled quantitatively to VLP's.

For the electron microscopic analysis, 10 ul of the protein solution on a

Formwar and coal-coated copper grid adsorbed, washed several times with water and then colored with 2% uranyl acetate.

Figure 1A shows the measurement of the sedimentation rate by means of analytical

Ultracentrifugation. Figure 1B shows an electron micrograph of the

VLP's.

Example 2: Packing of DNA by in vitro assembly of VPl To assemble into VLPs while simultaneously packing dsDNA, 1.98 μM pentameric VPl (0.42 mg / ml) with 8.42 nM dsDNA with a length of 100 bp, 184 bp or 300 bp were used incubated for 30 minutes at 25 ° C. To form the VLPs, these approaches were used against assembly buffer (50 mM Tris, 50 mM NaCl, 5% glycerol (v / v), 25% D-sorbitol (w / v), 2 mM CaCl ₂ , 4.5 mM GSSG, 0 , 5 mM GSH, pH 7.4) dialyzed for 48 h at 24 ° C. In order to demonstrate the packaging of DNA in the VLPs obtained, the samples were first dialyzed for 24 h at 8 ° C. against a sorbitol-free buffer (50 mM Tris, 50 mM NaCl, 5% glycerol (v / v), 1 mM CaCl ₂ , pH 7.4). 200 μl of this sample were then subjected to a DNase digestion (400 units of Merck's benzonase) in order to avoid packaging To break down DNA fragments. 5 mM Mg Cl ₂ and 400 U bezonase (Merck) were added and incubated for 1 h at 37 ° C. In control experiments it could be shown that free DNA, as well as DNA fragments bound to pentamer or non-specifically to VLP's, are completely degraded. In order to inactivate the benzonase and to expose the DNA fragments in the capsids, 50 μl of a stop mix (0.5 M NaH PO, 0.5 M EDTA, 1 M DTT, pH 6.0) were added and for Incubated for 30 min at 25 ° C. For denaturation, the samples were then boiled at 95 ° C. for 15 minutes. To isolate the DNA fragments, a phenol-chloroform extraction and then a concentration using the GFX ™ PCR Kit was carried out. The packaged fragments could be displayed by agarose gel electrophoresis (2% agarose) and subsequent ethidium bromide staining.

Figure 2 shows the results of the DNA packaging described here. 100 bp, 180 bp and 300 bp dsDNA fragments were packaged. An unassembled pentamer / DNA sample served as a control. Lanes 4, 7 and 10 show the approaches under native conditions. Lanes 5, 8 and 11 under denatured conditions. Lanes 6, 9 and 12 show samples of the concentrated DNA after the benzonase digestion procedure, i.e. DNA packaged in wtVPl capsid.

Example 3: Detection of the binding of double-stranded DNA (dsDNA) to VP 1

A gel shift assay was used to detect dsDNA bound to pentameric Polyoma VPl. 10 μg VPl were incubated with 0.5 μg DNA of an 1kb or 100 bp conductor from NEB at 25 ° C. for 30 minutes. The samples were then mixed with glycerol and analyzed by agarose gel electrophoresis (1.8% agarose) and ethidium bromide staining. Figure 3 shows the running behavior of the lkb ladder dsDNA in the presence (lane 1) and absence of VPl (lane 2). The running behavior of the lOObp ladder dsDNA is also shown in the presence (lane 3) and absence of VPl (lane 4). In both cases it becomes clear that the binding of the DNA to Polyoma VPl changes its running behavior.

Example 4: Quantification of the binding of dsDNA to Polyoma VPl

The fluorescence polarization method was used to quantify DNA binding. 23 nM of a Rhodamine-labeled DNA fragment of 184 bp in length was also analyzed

VPl titrated. After each addition of the protein to the DNA, the sample was kept for 10 minutes

Incubated 20 ° C and then measured the polarization signal. For this, the

Fluorescence excited at 587 nm and the emission detected at 604 nm.

Figure 4 shows the change in the polarization signal of the labeled DNA with increasing

VPl concentration (•). This dependency can be described mathematically as education

of a VPl-DNA complex with a 1: 1 stoichiometry and a dissociation constant of Kd = 103 nM (-). (A) Linear plotting. (B) Double-reciprocal application.

Example 5: Influence of ionic strength on the binding of dsDNA

175 nM pentameres Polyoma VPl, dissolved in 50 mM Tris, pH 7.4, 1 mM EDTA, 5% glycerol, was incubated for 30 minutes at 20 ° C. with 250 nM plasmid DNA at different concentrations of NaCl. The amount of DNA-bound VPl was then determined by means of analytical ultracentrifugation. For this purpose, the samples were centrifuged at 40,000 rpm, 20 ° C. and the sedimentation behavior of the free and bound protein was determined at 10-minute intervals by measuring the absorption of the samples at 230 nm. Figure 5 shows the proportion of DNA-bound VPl as a function of the NaCl concentration of the buffer. It becomes clear that the binding of VP1 to DNA is cooperatively suppressed at NaCl concentrations above 80 mM.

Example 6: Detection of the binding of single-stranded DNA (ssDNA) to VPl. A gel shift assay was used to detect ssDNA bound to pentameric Polyoma VPl

used. 10 μg VPl were mixed with 2 μg φX174 virion DNA from NEB at 25 ° C. for 30

Minutes incubated. The samples were then mixed with glycerol and analyzed by agarose gel electrophoresis (0.7% agarose) and ethidium bromide staining.

Figure 6 shows the running behavior of the φX174 Virion DNA in the presence (lane 1) and

Absence of VPl (lane 2,3). Lane 4 represents a molecular weight standard. It becomes clear that the binding of the DNA to Polyoma VP1 changes its running behavior.

Example 7: Detection of the binding of single-stranded RNA (ssRNA) to VPl. A gel shift assay was used to detect ssRNA bound to Pentameres Polyoma VPl. 10 μg VPl, which had previously been mixed with SUPERaseln RNase inhibitor from Ambion for 10 minutes, were incubated with 1.5 μg of the RNA ladder from NEB at 25 ° C. for 30 minutes. The samples were then mixed with glycerol and analyzed by agarose gel electrophoresis (1% agarose) and ethidium bromide staining. Figure 7 shows the running behavior of the RNA molecular weight ladder in the presence (lane 1) and absence of VPl (lane 2.3). It becomes clear that the binding of the RNA to Polyoma VPl changes its running behavior.

Claims

claims

1. Process for the production of virus-analogous particles by in vitro assembly of capsomers or assembly of capsomers, to whose charged amino acids biologically active macromolecules are bound, with simultaneous packaging of the macromolecules in the presence of non-ionic stabilizers.

2. The method according to claim 1, characterized in that the capsomers or capsomers, to which biologically active macromolecules are bound, are assembled in a buffer with an ionic strength of <250 mM in the presence of an oxidizing redox system.

3. The method according spoke 1 and 2, characterized gekennzeiclmet that dsDNA, ssDNA, dsRNA, ssRNA, PNA, ribozymes, DNAzymes, RNAi, peptide- or protein-coding DNA, peptides or proteins are used as biologically active macromolecules.

4. The method according to claim 1 to 3, characterized in that sugar compounds of the classes of C3- C4-, C5-, C6- compounds are used as stabilizers as monosaccharides or oligo- or polysaccharides or other polyols such as ethylene glycol or polyethylene glycol. Method according spoke 1 to 3, characterized in that thiol-containing substances are used in their reduced and oxidized form as the oxidizing redox system.