HK1024714B - Dumbbell expression constructs for gene therapy - Google Patents

Description

The invention relates to a design principle for a minimal expression construct which, in addition to promoter and termination sequences necessary to control expression, does not contain any genetic information other than that to be expressed.

The design principle is intended to be used for the design of expression constructs for the expression of MHC-I or MHC-II presentable peptides, cytokines or components of cell cycle regulation, or for the synthesis of regulatory RNA molecules such as antisense RNA, ribozymes or mRNA-editing RNA. Furthermore, an essential part of the invention is that the design principle allows covalent linkage of the expression construct, for example with peptides, proteins, carbohydrate or glycopeptides, and particles that can be used for ballistic transfer of the constructs into cells, in particular the dermis, muscle, pancreas and liver.

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Laboratory experiments and clinical trials using such peptides to induce or enhance a tumour-specific cytotoxic response have focused on conventional vaccination protocols using the respective peptides (Strominger J., Nature Medicine, (1995) 1: 1,179-1,183). Alternatively, antigen-presenting cells, such as dendritic cells, have been incubated with high concentrations of such peptides, thereby exchanging the peptides originally presented by the MHC complex for the tumour-specific peptides (Grabbe et al., Immunology Today (1995) 16:117-121).

The term genetic vaccination describes the use of an experimental finding that was initially controversial scientifically as an artifact but has recently been confirmed for a variety of biomedical issues (Piatak et al., Science 259 (1993): 1745-1749). When an expression plasmid is injected into mammalian cells in skin or muscle layers, it is, albeit with very low efficiency, injected into the cells at the site of injection to express the corresponding gene. If the expression product is a foreign protein (xenogenic or allogeneic protein), it is likely to result in the uptake and presentation of antibodies during a localized inflammation, which is the expression of the antigenic gene (Human Peptide Transcriptase) (HTP4O1 or MTP4O2), which is also expressed in vivo via the human immunoglobulin gene (Human Peptide Transcriptase) (HTP4O2 or MTP4O2), which is also known locally as the antigenic antigenic drug (THP4O2 or MTP4O2) and is used in this product.

This genetic vaccination avoids the multiple risks of conventional immunization methods. Numerous approaches are known in gene therapy to achieve therapeutic or prophylactic effects by introducing genetic information into cells. These approaches have been demonstrated not only in animal studies but also in numerous clinical studies on patients, for example the so-called ballistic magnetic vector system (EP 0 686 697 A2) for transfection with conventional plasmid-based expression constructs. The ballistic magnetic vector system was used by the local medicine communicator/inventor in three clinical phase I/II studies for the production of unprepared gene constructs for IL-7 (IL-7), IL-12 (IL-12-12) or Granulocytocol and other macromolecular tumor-causing factors (GMF-1144-1, IL-12-46) and two simultaneous transfections of the two molecules (IMM-1244-1, IL-12-46) of the same expression.

The new discipline of gene therapy, however, requires a critical examination of the existing catalogue of gene therapy methods. An essential aspect is the sequence information contained in the DNA constructs used so far. If expression constructs are to be used in large numbers of patients, and possibly several times, safety aspects will gain a lot of weight, especially from an immunological point of view. The expression constructs used so far are further developments of eukaryotic expression plasmids. These have two fundamental disadvantages: their size, which prevents rapid transport into the cell nucleus, and the presence of sequences not necessary for the actual application.

It is obvious that the present expression constructs not only lead to the expression of the desired gene but also to the biosynthesis of xenogenic proteins, even if their prokaryotic promoters have very low activity in mammalian cells.

Another problem in the application of gene therapy methods is the method of introducing the genetic material to be transferred into cells. For reasons of efficiency, immunological safety and wide applicability across many cell types, the method of ballistic transfer is preferred. A major advantage of ballistic transfer over alternative transfection methods is that the method is relatively easily transferable between different cell or tissue types.The most important factor in the development of the cell is the ability of the cell to produce a new nucleic acid, which is the most important factor in the development of the cell, and the ability of the cell to produce a new nucleic acid, which is the most important factor in the development of the cell.For many slowly dividing or not dividing cell types which are of interest in the context of possible gene therapy, such as stem cells of the immune system or haematopoiesis, muscle cells, cells of exocrine or endocrine secretory organs, or neurons and their accompanying cells, these methods are therefore not suitable.

The method of ballistic transfer has been used in the ex vivo treatment of autologous or allogeneic patient cells (Mahvi et al.; Human Gene Therapy 7 (1996): 1535-43). However, in the treatment of cells in the cell band, which would be particularly advantageous for the oncotherapeutic treatment of solid tumors or the prophylaxis of mass infections by genetic vaccination, the state of the art has disadvantages: the method of ballistic transfer uses the adsorbed DNA microprojectiles.In the case of solid tumour tissue (colon, rectal, renal and other cancers), it was found that, when the parameters were adjusted appropriately, the micro-projectiles reached a depth of penetration of up to five cell layers into the tissue sections, but the transposed cells (visible as fluorescent cells when transposed with a recombinant expression constructor containing the ae-structural gene for the green fluorescent protein Proquorea Victoria) were all located at the top, just above the surface of the head, which was fixed to the projectile vessel, which was directed towards the head of the DNA.The first is to use a new method of gene therapy to prevent the transfer of the substance to be transported. Only in this way is it realistic to apply gene therapy to solid tumours, since only transfection of tumour sections deep in the tissue allows a sufficiently large number of treated cells to be transferred. At the same time, it is conceivable that a combination of different coupling methods could allow the release of multiple genetic information within the same cell population in a time-delayed manner.

US 5,584,807 (McCabe) describes an instrument in the form of a gas-pressure gun for introducing genetic material into biological tissue, using gold particles as a carrier material for the genetic information, but without going into the genetic material in particular. US 5,580,859 and US 5,589,466 (Felgner) describe a procedure for introducing DNA into mammalian cells as part of gene therapy. This involves injecting so-called naked DNA sequences encoding physiologically active proteins, peptides or polypeptides and equipped with a promoter directly into cells. The DNA is expressed in the cells and serves as a vaccine.

WO 96/26270 (Rhone-Poulenc Rorer S.A.) describes a circular double-stranded (supercoiled) DNA molecule containing an expression cassette, coding for a gene and controlled by a promoter and terminator.

EP 0 686 697 A2 (Soft Gene) concerns a method of enrichment of cells modified by ballistic transfer and describes the technological background and the problems involved and their known solutions to date.

The ballistic particles (EP 0 686 697 A2) are gold particles with a diameter of either 1 μm or 1.5 μm, selected depending on the cell type. The gold particles are coated with superparamagnetic particles of approximately 30 nm in diameter. The superparamagnetic particles also provide a suitable surface for coating with biomolecules. The use of magnetic particles is used for sorting.

Hump-shaped nucleic acid constructs are also known, characterised by the following characteristics: they are short (10-50 base pairs of double-stranded DNA) nucleic acid constructs produced for structural research or as a double-stranded, nuclease-based oligomer for sequence-specific DNA ligands (Clusel et al.; Nucleic Acids Res. 21 (1993): 3405-11; Lim et al., Nucleic Acids Res. 25 (1997): 575-81).

Longer DNA molecules, which may also be present as dumbbells during part of their replication cycle, are known in nature as mitochondrial genomes of some species, such as cilia and yeast (Dinuel et al., Molecular and Cell Biology, 13 (1993): 2315-23). These molecules are about 50 kb in size and have a very complex genetic structure.

Peptide nucleic acid linkages with localization sequences are known for short DNA oligomers. Morris et al. (Nucleic Acids Res. 25 (1997): 2730-36) describe the coupling of 18-mer to 36-mer with a peptide containing 27 amino acid residues containing the localization sequence from SV40 and a signaling peptide from HIV-gp41 responsible for fusion with CD4-positive cells.

The use of peptide chains to cross the endosomal membrane has been investigated by several groups, for example, the 23 N-terminal amino acids of haemagglutinin were adsorbed via non-covalent interactions to expression plasmids to bind these expression plasmids to the cytosol after endosomal uptake of the complexes (Plank et al., J. Biol. Chem. (1994) 269: 12918).

The present invention is intended to develop an expression construct which contains only the information necessary for expression and to provide suitable means of transport into a cell to be treated with gene therapy.

This task is solved by the features of claims 1 and 13. The invention modifies the double-stranded deoxyribonucleic acid expression constructs to be transported so that the antiparallel strands of the deoxyribonucleic acid polymer containing the coding sequence and the promoter and termination sequences necessary for their expression are linked by a loop of single-stranded deoxyribonucleotides at each end, covalently linking to form a highly covalently closed RNA molecule. Preferably, this loop is very long, up to three molecules. Figure 1 shows a similar structure. The structure of this interlock is used to maintain intracellular expression of the active substance, such as C-HPC-I or M-RNA, and thus to regulate the expression of the active substance in the body, such as MHC-II or MHC-II, and to maintain a stable and stable structure in the body.

Furthermore, according to the invention, the loop connecting the strands may contain one or more modified bases containing chemical functions which enable them to be coupled to a fixed phase, preferably amine, carbonic acid, thiol or disulfide modifications, which are covalently linked to the gold surface of a ballistic micro-projector by known synthesis steps with corresponding carbonic acid, aldehyde, amine, thiol or other functions or directly to the gold surface of a ballistic transfer.

In addition to the advantage of facilitating chemical binding to the surface of the micro-projector, the aforementioned nucleic acid construction also offers another advantage: the nucleic acid constructs used today for transfection in gene therapy are produced and carried in bacteria, in addition to the sequences relevant in the context of therapeutic use, also sequences that are important only for the multiplication of the nucleic acid structure in the bacteria. These sequences pose an unknown risk to the patient to be treated, since it is not known whether and if necessary, they can be modified by the target organism. They therefore contain solenoid sequences that do not exclusively alter the primary expression of the transfection in the target cells, polymers that are used in the bacterial transfection to induce the nucleic acid expression by removing the residues of the chemical.

The present invention is further supported by the fact that chemical modifications can be made to the single-stranded deoxyribonucleotide loop at either end of the molecule to covalently link non-nucleic acid ligands to the nucleic acid expression construct, such as the ability of peptides leading to the nuclear localization of the expression construct to bind to the construct in such a way that, even after penetration, the latter is only carried into the cytosol of a cell from its translocation apparatus into nuclear compartments, where the translocation structures can be read off. This would remove the above-mentioned restrictions for some transfection constructs.

In particular, the double-stranded antiparallel expression constructs of deoxyribonucleic acid to be transported according to the invention are modified to contain at each end of the double-strands a disulfide bridge which links the strands at each end to each other via an alkyl group covalently bound at the 5' end of one and the 3' end of the other strand.

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The present invention also relates to the transport of nucleic acids into cells. Nucleic acids are introduced into cells by means of adsorption, covalent or ionic interactions, whereby the nucleic acids are bound to the surface of the micro-projector which brings the nucleic acids into the cells, so that when the micro-projector passes through connective tissue, the fluid or cell layers surrounding the cells, they are not or not completely desorbed but remain bound to the micro-projects until they come to rest with them in the target cells.

In general, the binding of thiols or disulfides to gold surfaces has been well studied and described (G. M. Whitesides et al., Langmuir 10 (1994): 1825-1831). However, the nucleic acids to be transported are preferably bound to the gold micro-projectile by first covalently modifying the nucleic acids with commercial reagents containing thiol or disulfide groups and then adsorbing them through the thiol or disulfide groups, or by anodic oxidation of the thiol or disulfide function using the gold micro-projectile as a sufficient covalent anode. In particular, the transporting substances are first covalently modified by covalent modification with commercial reagents containing thiol or disulfide groups and then adsorbed through the thiol or disulfide groups, or by anodic sub-oxidation of the thiol or disulfide function using the gold micro-projectile as a covalent anode. In particular, the transporting substances are bound to the gold by means of gold-bonded or disulfide-bonded compounds. The gold-bonded or disulfide-bonded micro-projectile is then bound to the gold by means of a highly modified or non-modifiable thiol or disulfide group.

Since the cell contains molecules that contain thiol groups, especially the ubiquitous glutathione, a slow desorption of the chemosorbent molecule from the surface of the micro-project occurs in an equilibrium reaction of the thiol groups on the gold surface, so that the transported substance is freely available to the cell after desorption. Furthermore, according to the invention, when using gold micro-projects, the molecule to be transported, which has thiol or disulfide bridges, can be bound to the micro-project by anodic oxidation of the thiol or disulfide function using the gold micro-project as an anode covalent.

The invention also provides that, when using micro-projects made of oxide ceramics, glass ceramics or glass, the molecule to be transported is bound by ester, amide, aldimin, ketal, acetal or ether bonds or other functionalities known to the organic chemist for binding molecules to solid surfaces.

The invention is intended for use in ex vivo gene therapy. Preferably, interleukin-7 (IL-7) and interleukin-12 (IL-12) proteins or their subunits are expressed, as well as interleukins, granulocyte macrophage colony stimulating factor (GM-CSF), surface antigens and ligands of immune-controlled or stimulating lymphocyte antigens such as CD40, B7-1 and B7-2, MHC complex I or II proteins, as well as beta-2 microglobulin, interferon-conjugate sequence-binding protein ICSBP, CI, FLITA, FLt3, and fragments or whole of these proteins, which can be constructed into a tumor-specific expression or are not distinct from each other, but are all constructed from a single or multiple Ki-FASR, p16 or b3, or from a single or multiple co-designed, but similar, microparticle, but are all constructed from a single or multiple co-designed, or a combination of the same or multiple co-designed, but are all constructed from a single or multiple microparticle, or a single product, and are also used for the same purpose.

The deoxyribonucleic acid construct of the invention is also of preference for use as a vaccine for the treatment of infectious diseases in humans and animals, such as malaria and influenza.

The other subclaims contain further advantages, the invention is shown in the accompanying drawings and described in more detail below by means of examples. Figure 1a schematic representation of the construct concept, where Figure 1.1 shows the structure of the covalently closed phosphate sugar chain; the number of base pairs shown does not necessarily correspond to the length of the constructs but is only for the purpose of clarification; Figure 1.2 shows the functional structure of one of several possible expression constructs;Figure 2 shows the schematic sequence of the construct representation.

Example 1 [Synthesis of the expression constructs]:

The basic design principle is shown in Figure 2 and is as follows:

1.1 Summary from Vector:

The gene to be expressed, e.g. granulocyte macrophage stimulating factor gm-csf, is amplified from cDNA by means of a suitable primer by PCR (Fig. 2 (1)) and recombined into a suitable plasmid vector (Fig. 2 (2)). After sequencing and confirming the target sequence, the sequence to be expressed from the said plasmid vector is amplified by means of two primers (oligodesoxynucleotides, which have recognition sites for restriction endonucleases on the 5' side of their sequence) (Fig. 2 (3)). The resulting amplification product is amplified with the said endonucleases, for which a recognition sequence of the said endonucleases was available. The expression of this isotopic is expressed in the desired isotopic isotope in the agarose (Fig. 2 (4)), which is located in the primary and is expressed in the recombinant isotopic in which the expression is expressed in the desired isotope.

The expression plasmid is multiplied in bacteria and isolated by known methods. After digestion with restriction endonucleases, whose recognition sequences are flanked by the sequence to be carried by the funnel-shaped expression construct, the restriction fragments are isolated separately from each other by appropriate methods using anion exchange chromatography (Fig. 2 (5)) and then with hairpin-shaped self-hybridising oligode-deoxynucleotides (short DNA-moles produced by automated chemical DNA ligase synthesis, which can form stem-molecular structures due to their self-completion structure; these molecules later form the covariates of the closed end-module-shell anion exchange) which are removed by means of a duplicate structure of the DNA-molecule residues (Diplomerate-molecule-molecule) which are located on the surface of the DNA structure.

1.2 Synthesis from PCR product

Alternatively, the construct is directly multiplied by polymerase chain reaction, with the primary oligodesoxynucleotides on the 5' side of their sequence bearing detection sites for restriction endonuleases (Fig. 2 (7)). After ion exchange chromatographic separation of the primer, the resulting amplification product is digested with the endonucleases for which a detection sequence was present on the said primers. After further separation of the smaller restriction fragments, the construct is composed of short hairpin-shaped self-hybridizing oesoxynucleotides which are ligated to the restriction-digested residual structure of the construct by means of their superposition rings (Fig. 2 (8)).

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The following is the list of the active substances: Ballistic transfer of gm-csf to K562:

30 μl of a 1+3 mixture of a suspension of colloidal magnetic particles (medium diameter: 65 nm; Miltenyi GmbH, Berg-Gladbach, Germany; concentration of suspension: 30 mg/ml) is pipetted onto a macrocarrier polymer plate (Fa. Bio-Rad) and a gmcs rock-expression helical structure (P. Bio-Rad) is filtered. The gold is filtered off and the excess is carefully removed. 30 μl of a 1+3 mixture of a suspension of colloidal magnetic particles (medium diameter: 65 nm; Miltenyi GmbH, Berg-Gladbach, Germany; concentration of suspension: 30 mg/ml) is placed on the wet surface and the gold is filtered off. The gold is filtered off at a temperature of 300 μm.

The ballistic transfer is carried out on a Biolistic PDS 1000/He (Bio-Rad, Hercules, CA, USA) as prescribed by the manufacturer of the apparatus. The burst disc used corresponds to a set breaking pressure of 1,100 psi. The vacuum cell pressure is 508 mm mercury column (20 in Hg). Magnetic separation is carried out as published (EP 0 732 395 A1); success control is done by gm-csf-ELISA.

Example 3 [Example of making an expression construct with nuclear location sequence]:

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Example 4 [synthesis of nucleic acid modified gold particles]:

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Example 5 [Ballistic transfer to solid tumour tissue]:

Necrotic and connective tissue parts are removed as far as possible. Pieces of about 1 cm2 are cut out, washed in ice-cold PBS and fixed with tissue adhesive on the sample holder of a tissue sectioning system (vibratome 1000 sectioning system; TPI, St. Louis, Missouri). The tumor is cut into 500 μm-thick slices. The slices are stored in ice-cold PBS and transfected as quickly as possible. 30 μl of a suspension of gold-expression gold-coated gold-rock-coated articles and colloidal magnetic plates (through a 65 nm transistor; the transistor is passed through the two-layered polymer-transparent membrane) are described as two identical slices (see Figure 2), and the process is described by the following steps:

Magnetic separation is carried out as published (EP 0 732 395 A1); success control is carried out by GM-CSF-ELISA.

The following is the list of the active substances in the active substance:

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1 g of aluminium oxide particles (medium diameter 1.0 μm) were boiled overnight in a solution of triethoxyaminopropyl silane in toluene (2%) at the return stream. The solid is filtered, washed with toluene, then ethanol, dried and ground again. 5 mg of the resulting amino-modified aluminium oxide is placed in 100 ml of aqueous carbonate buffer (pH 8.0) with 4 μg of the carbonate modified construct in the presence of 50 mM 1-ethyl-3-dimethypropylmid) carboxylamide and 50 mM N-hydroxysuccinamide at 2 °C. The resulting nucleic acid can be rapidly converted into cells by accelerated oxidation. The information is then passed to the transport chamber in a device such as the EP 396 and the A102A1A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A

Claims (18)

1. Deoxyribonucleic acid construct for transcription of RNA-molecules, characterized by a circular strand of deoxyribonucleic acid comprising a partly complementary, antiparallel base sequence, so that a dumbbell-shaped construct is formed,

   - in which the complementary, antiparallel base sequence in the essential comprises a promotor sequence, a coding sequence and a polyadenylation signal or another RNA-stabilizing signal,

   - and the non-complementary sequence comprises two loops of single-stranded deoxyribonucleic acid, which covalently join the 5'- and 3'- ends of the complementary, antiparallel strands.
2. Deoxyribonucleic acid construct according to claim 1, characterized by that said loops consisting of three to seven nucleotides, and in which one or several of said nucleotides are covalently modified by carboxylic acid-, amine-, thiole- or aldehyde functionalities.
3. Deoxyribonucleic acid construct according to claim 2, characterized by that said chemically modified nucleotides is being linked to a peptide leading to the directed transport of the construct into the nucleus.
4. Deoxyribonucleic acid construct according to claim 2, characterized by that said chemically modified nucleotides is being linked to a peptide enabling liberation of the construct from the endosome.
5. Deoxyribonucleic acid construct according to claim 1, characterized by using a 7SK promoter as said promoter.
6. Deoxyribonucleic acid construct according to claim 1, characterized by using a CMV promoter as said promoter.
7. Deoxyribonucleic acid construct according to at least one of the claims a to 6, coding for interleukine-7.
8. Deoxyribonucleic acid construct according to at least one of the claims 1 to 6, coding for interleukine-12 or one or several of its constituting subunits.
9. Deoxyribonucleic acid construct according to at least one of the claims 1 to 6, coding for gm-csf.
10. Deoxyribonucleic acid construct according to at least one of the claims 1 to 6, coding for p16 or p53 protein or fragments thereof.
11. Deoxyribonucleic acid construct according to at least one of the claims 1 to 6, coding for peptide fragments of mutated ki-ras, mutated p53 or bcr-abl translocation product with a length of between 10 and 100 amino acids.
12. Micro projectile for ballistic transfer of deoxyribonucleic acid constructs into cells according to one or several of claims 1 to 11, in which the substance to be transported is linked by adsorption or covalent or ionic binding in such a way to said micro projectile that the substance to be transported upon passage of the micro particle through connective tissue, the extra cellular liquid or cell layers is not or not completely desorbed, but remains bound to said micro projectile until the substance to be transported rests along with said micro projectile in the target cell.
13. Micro projectile according to claim 12, characterized by that its material being gold, micro crystalline gold, oxide ceramic, glass ceramic or glass and said nucleic acid to be transported is bound covalently by thiole- or disulfide moieties, ester-, amide-, aldimine-, ketale- or acetale- or ether functionalities to said micro projectile.
14. Micro projectile according to claim 12 or 13, characterized by that said micro projectile being made out of an electrically conducive material and said nucleic acid being linked to said micro projectile by electrochemically coupling of disulfide or thiole moieties, employing the micro projectile as electrode.
15. Micro projectile according to claim 12 to 14, characterized by that said micro projectile being of the size of 0,3 µm to 3 µm.
16. Use of a nucleic acid construct according to one or more of the claims 1 to 11 in ex-vivo gene therapy.
17. Use of a micro projectile according to one or more of the claims 12 to 15 in ex-vivo gene therapy.
18. Use of said deoxyribonucleic acid constructs according to one or several of the claims 1 to 6 for producing a vaccine for prevention of infectious disease in humans or animals.