Patent Application
20210205471
The present invention relates to the medical field of monogenetic disorders, in particular monogenetic disorders associated with mutations in genes coding for proteins expressed for example in the liver.
In particular, the present invention relates to conjugated gold nanoparticles, preferably for the use in the treatment of a monogenetic disorder resulting from a mutation in a gene coding for a liver-specific and/or liver-expressed protein, and the respective use of the particles.
Furthermore, the present invention relates a method for the preparation of conjugated gold nanoparticles, a nanoparticle-based delivery system and the use of the respective delivery system. A further subject of the present invention relates to a method for transfection of target cells and transfected target cells as such. Finally, the present invention relates to a vector to be used in the gold nanoparticles according to the present invention.
The liver is a vital organ of the human body and has a wide range of functions, including the detoxification of various metabolites, protein synthesis and the production of biochemicals necessary for digestion. Furthermore, the liver plays a central role in metabolism, regulation of glycogen storage, decomposition of red blood cells and hormone production.
As outlined before, one main function of the liver is the production of proteins and their subsequent secretion into the blood. Proteins produced and secreted by the liver include major plasma proteins, carrier proteins, hormones, prohormones and apolipoproteins. In particular, the liver produces and secretes proteins and factors, which regulate hemostasis, i.e. blood clotting.
Furthermore, the liver produces and secretes proteins involved in lipometabolism, amino acid metabolism, bilirubin metabolism, urea cycle metabolism, carbohydrate metabolism, proteoglycan metabolism and sphingolipid metabolism. Additionally, the liver produces the antiprotease alpha-1-antitrypsin as well as proteins involved in transportation processes.
Hemostasis occurs when blood is present outside of the body or blood vessels. During hemostasis three steps occur in a rapid sequence. The first step includes a vascular spasm or a vasoconstriction, respectively. By vasoconstriction, the amount of blood flow can be reduced and the blood loss can be limited. Furthermore, collagen is exposed at the site of injury, thereby promoting platelets to adhere to the injury site. The second step of hemostasis includes the formation of platelet plugs.
Thereby, platelets adhere to the damaged endothelium to form a plug. This process is also called primary hemostasis. Once the plug has been formed, clotting factors begin creating the clot. Thereby, the clotting factors begin to form fibrin factor (FIa). Fibrin is a fibrous, non-globular protein, which is formed by the action of the protease thrombin factor (FII). This third step of hemostasis including the coagulation is also called secondary hemostasis. Thereby, the platelet plug is reinforced, wherein fibrin threads function as glue for the sticky platelets.
A multitude of factors and proteins is involved in the secondary hemostasis, for example fibrinogen (FI), prothrombin (FII), tissue factor/tissue thromboplastin (FIII), calcium (FIV), proaccelerin (FV), proconvertin (FVII), antihemophilic factor A (FVIII), antihemophilic factor B (FIX), Stuart-Prower factor (FX), plasma thromboplastin antecedent (FXI), Hageman factor (FXII) and fibrin-stabilizing factor (FXIII), wherein the list of factors is not exhaustive with respect to factors and proteins regulating hemostasis.
A diminished or absent production of blood clotting factors can lead to a phenotype or disease called hemophilia. Hemophilia is a term for a group of blood clotting disorders whose clinical symptoms are caused by a diminished or absent activity of blood clotting factors. Hemophilia is a mostly inherited in particular monogenetic disorder that impairs the body's ability to make blood clots, a process needed to stop bleeding. People suffering from hemophilia usually bleed longer after an injury and bruise easily. Furthermore, the disorder leads to an increased risk of bleeding inside joints or the brain.
The two most common subforms are hemophilia A with an incidence of 1:10.000 due to loss-of-function mutations in the gene coding for coagulation factor FVIII and hemophilia B with an incidence of 1:50.000 due to mutations in the factor FIX gene. Hemophilia A and B are caused by inherited and also de novo mutations in the X-chromosomally localized FVIII and FIX genes, which lead to loss of protein activity and thereby interfere with the coagulation cascade causing severe bleeding episodes. Because of the X-chromosomal recessive inheritance, almost exclusively boys and men are affected, while females as heterozygous germ-line mutation carriers show a reduction of the factor activity measurable in the laboratory, but are clinically healthy, i.e. without symptoms. Based on the residual activity of FVIII or FIX in the plasma, severe (less than 1% activity), moderate (1 to 5% activity), mild (6 to 24% activity) and subhemophilia (25 to 50% activity) can be distinguished. Notably, more than 50% of patients are affected by severe hemophilia. Patients with severe and also moderate hemophilia suffer about 30 to 40 severe bleeding episodes per year. Bleeding occurs spontaneously or after slight trauma. Mild and subhemophilia are clinically apparent only after surgery, trauma or treatment with acetylsalicylic acid or related drugs.
The WHO currently estimates that the number of patients worldwide is >400.000, of which approximately 10.000 hemophils are living in Germany. The current therapy for clinically severe moderate hemophilia involves a regular prophylactic use of concentrated FVIII or FIX products by intravenous injections. This prophylaxis allows an almost normal life expectancy and quality of life for hemophilia patients. According to the scientific publication of Oldenburg: “Optimal treatment strategies for hemophilia: achievements and limitations of current prophylactic regiments”, published in Blood, 2015, 125(13):2038-44, in context with prophylactic treatment of hemophilia, concentrated FVIII or FIX products are either isolated as plasmatic factors from healthy blood donors or recovered as recombinant factors from specific cell cultures. A regular prophylaxis prevents long-lasting clinical consequences of the bleeding episodes including disabilities due to intracranial hemorrhage and chronic joint diseases and musculoskeletal crippling problems. Disadvantageously, the prophylactic treatment generates very high costs per year for each patient to be treated. Furthermore, the recurring treatments are rather stressful for the patients. Moreover, according to Peyvandi et al.: “A randomized trial of factor VIII and neutralizing antibodies in hemophilia A”, published in N. Engl. J. Med., 2016, 374(21):2054-64, more than 50% of patients with severe hemophilia do not produce any endogenous FVIII or FIX. In this patients, administration of the exogenous proteins results in the development of neutralizing antibodies, so-called inhibitors, in up to 45% of the cases. These inhibitors neutralize the substituted factors and thereby render the factor replacement therapy ineffective. In patients with inhibitors, immune tolerance induction can be achieved by treatment with high doses of factors over a period of one to two years. However, this approach is only successful in 50 to 70% of patients. Additionally, the immune tolerance induction leads to a significant increase of costs per patient per year.
Since hemophilia is—in the majority of cases—a monogenetic disorder, multiple efforts to treat the disease with different gene therapy strategies have been pursued. The basic goal of all gene therapy approaches is the permanent introduction of an intact copy of the defective gene as complementary DNA (cDNA) into the nucleus of the target cell.
Recombinant gene delivery systems for the intact gene are so-called vectors, which are mostly derived from viral systems. These wild-type viruses are evolutionarily optimized in terms of their properties to efficiently transfer their genetic information to the target cell and into the nucleus of the cell, respectively.
The viral gene transfer system most frequently used for hemophilia originates from the adeno-associated virus (AAV), which exists in various different serotypes and can infect primary liver cells particularly well. The use of an AAV-based gene transfer system has been described by High and Anguela: “Adeno-associated viral vectors for the treatment of hemophilia”, published in Hum. Mol. Genet., 2016, 25(R1):R36-41. In addition, lentiviral vectors derived from the human immunodeficiency virus (HIV-1) have been used and can very efficiently integrate into the DNA of dividing and also non-dividing cells. In all these viral approaches, the integration of the vector DNA into the genome of the target cell appears to be the greatest risk. Here, the function or expression of a gene located in the vicinity of the insertion site can be altered or modified by the integration event and thus can lead to a malignant transformation of the cell.
Another viral approach on the basis of a gene therapy for hemophilia B with an AAV-FIX vector was described by Nathwani et al. in the scientific publications “Adenovirus-associated virus vector-mediated gene transfer in hemophilia B”, N. Engl. J. Med., 2011, 365(25):2357-65, and “Long-term safety and efficacy of factor IX gene therapy in hemophilia B”, N. Engl. J. Med., 2014, 371(21):1994-2004. An important side effect or a severe adverse event, respectively, of the therapy was an increase of the liver enzymes. The liver toxicity required an additional cortisone therapy. Furthermore, patients once treated with a specific AAV serotype will develop lifelong immunity to the specific AAV envelope protein and can never be treated with the same vector or serotype again.
Similarly, a concept for a gene therapy for hemophilia A on the basis of an AAV-FVII vector has been developed. According to the scientific publication of Nault et al. “Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas”, Nat. Genet., 2015, 47(10):1187-93, a therapy on the basis of wildtype AAV might be linked with the risk of developing hepatocellular carcinoma in humans.
Moreover, non-genetic approaches for the treatment of hemophilia consist in the use of antibodies. In this context, for the treatment of hemophilia A, a bispecific humanized recombinant antibody has been described by Muto et al.: “Anti-factor IXa/x bispecific antibody ACE910 prevents joint bleeds in a long-term primate model of acquired hemophilia A”, published in Blood, 2014, 124(20):3165-71 as well as Kitazawa et al.: “A bispecific antibody to factors IXa and X restores factor VIII hemostatic activity in hemophilia A model”, published in Nat. Med. 2012, 18(10):1570-4. The respective antibody can replace the cross-linking of FIX or the active form FIXa, respectively, and FX as an essential function of FVIII in the coagulation cascade. Even though antibodies are not associated with the risk of mutagenesis, however, also a non-genetic therapy on the basis of antibodies can be linked with undesired side effects, in particular with respect to undesired immunological reactions.
Furthermore, with respect to gene therapy in general, efforts have been made with respect to the use of nanoparticles, such as chemically generated gold nanoparticles, as carrier for nucleic acids. In general, chemically generated gold nanoparticles are suitable to mediate a gene or DNA transfer to the target cells. Nevertheless, there use is linked with several adverse effects. On the one hand, in particular organic residues of the preparation process lead to a certain cell toxicity and undesired interactions between the particles, in particular agglomerations of particles. On the other hand, the loadability with transfection agents and genetic material is still not sufficient and linked with a reduced transfer of target DNA.
Overall, there is a strong need for improved therapeutic concepts and/or approaches with respect to the treatment of monogenetic diseases associated with mutations in genes coding for proteins predominantly expressed in the liver, in particular proteins of the coagulation cascade and/or proteins involved in hemostasis. Especially, there is a strong need for improved therapeutic concepts for the treatment of hemophilia.
Against the background of the severe disadvantages of known therapeutic concepts for the treatment of monogenetic disorders, in particular hemophilia, as delineated before, the problem of the present invention is based on the supply of a new therapeutic concept for the treatment of monogenetic disorders associated with mutations in genes coding for liver-specific and/or liver-expressed proteins and/or proteins predominantly expressed in the liver, in particular proteins involved in hemostasis and/or proteins or factors of the coagulation cascade.
In particular, the object of the present invention has to be seen in a therapeutic concept for the treatment of monogenetic disorders associated with the liver, especially hemophilia, on the basis of preferably non-viral gene therapy with an improved efficiency with respect to the transfer of genetic material as well as reduced side effects and a lowered cell toxicity.
The applicant has surprisingly found, that the aforementioned problem can be solved—according to the first aspect of the present invention—on the basis of a conjugated gold nanoparticles as claimed in claim 1; further, in particular advantageous embodiments of this aspect are subject-matter of the respective dependent claims.
Additionally, the present invention relates to—according to the second aspect of the present invention—the inventive use of the conjugated gold nanoparticles according to the respective independent claim; further, in particular advantageous embodiments of this aspect are subject-matter of the respective dependent claims.
Furthermore, subject-matter of the present invention is—according to the third aspect of the present invention—a method for the preparation of conjugated gold nanoparticles according to the respective independent claim; further, in particular advantageous embodiments of this aspect are subject-matter of the respective dependent claims.
The present invention also relates to—according to the fourth aspect of the present invention—a nanoparticle-based delivery system according to the respective independent claim; further, in particular advantageous embodiments of this aspect are subject-matter of the respective dependent claims.
In addition, subject-matter of the present invention is—according to the fifth aspect of the present invention—the use of a nanoparticle-based delivery system according to the respective independent claim; further, in particular advantageous embodiments of this aspect are subject-matter of the respective dependent claims.
Furthermore, the present invention relates to—according to a sixth aspect of the present invention—a method for transfection of target cells.
Another subject-matter of the present invention is—according to a seventh aspect of the present invention—a transfected target cell; further, in particular advantageous embodiments of this aspect are subject-matter of the respective dependent claims.
Finally, the present invention relates—according to an eighth aspect of the present invention—a vector for the expression of a liver-specific and/or liver expressed protein; further, in particular advantageous embodiments of this aspect are subject-matter of the respective dependent claims.
With respect to the aspects of the present invention it has to be pointed out that explanations, which have been made in relation to one aspect self-evidently also apply with respect to the other aspects.
Apart from this, a person skilled in the art can—depending on the application or depending on the individual case—deviate from the specified weights, specified quantities and specified ranges that are stated below without departing from the scope of the present invention.
Moreover, all specified values or specified parameters or the like that are mentioned below can absolutely be ascertained or determined using normed or standardized or explicitly specified determination methods or else using determination or measurement methods familiar per se to a person skilled in the art in this field.
With this said, the present invention will now be elucidated in detail below:
The present invention therefore provides—according to a first aspect of the present invention—conjugated gold nanoparticles, preferably for the use in the treatment, in particular a non-viral gene therapy, of a monogenetic disorder resulting from a mutation in a gene coding for a liver-specific and/or liver-expressed protein, comprising:
On the basis of the present invention conjugated gold nanoparticles have been developed which are suitable for the use in a novel non-viral gene therapy approach, wherein the transfection efficiency and/or the gene transfer efficiency is surprisingly improved on the basis of the use of laser-ablated gold nanoparticles. Furthermore, the use of laser-ablated gold nanoparticles is linked with a lowered toxicity and immunogenicity, when compared to the use of chemically synthesized gold nanoparticles. The conjugated gold nanoparticles according to the present invention are suitable for the transfer of intact copies of any coding sequence containing a nucleic acid sequence coding for a liver-specific and/or liver-expressed protein in order to allow the production of a therapeutically effective amount of the protein in the transfected cells.
In particular, the conjugated gold nanoparticles are suitable for the use in a novel gene therapy for hemophilia, allowing for a therapeutically effective production of the missing blood clotting factors in the patients, preferably coagulation factors VIII and/or IX. Nevertheless, the concept according to the present invention is suitable for the transfer of any other liver-specific and/or liver-expressed protein, in particular liver-specific and/or liver-expressed proteins which are associated with a monogenetic disorder.
The conjugated gold nanoparticles according to the present invention are linked with several advantages over known therapeutic concepts for the treatment of monogenetic disorders, in particular hemophilia:
According to the present invention it was surprisingly found that the use of laser-ablated gold nanoparticles as carrier material or carrier system is linked with several advantages when compared with known genetic approaches for the treatment of monogenetic disorders, in particular viral gene transfer systems, on the one hand, and chemically sympathized gold nanoparticles, on the other hand.
The use of gold nanoparticles obtained by laser ablation, in particular pulsed laser ablation in liquid, leads to an improved transfection efficiency, i.e. a higher transfection rate of target cells, as well as an efficient endosomal release of the nucleic acid molecules, in particular the vector, after cellular uptake. Furthermore, gold nanoparticles obtained by laser ablation are linked with a lesser toxicity and immunogenicity, in particular when compared to chemically synthesized gold nanoparticles. Overall, the use of gold nanoparticles obtained by laser ablation in the conjugated nanoparticles according to the present invention is safer when compared to approaches on the basis of viral vectors and chemically synthesized gold nanoparticles, on the one hand, and linked with an improved therapeutic efficacy, on the other hand. Furthermore, in contrast to chemically synthesized particles, undesired reactions, in particular the formation of agglomerates, can be prevented.
Without being bound to this theory, the advantages over chemically synthesized particles might be a result of the following physico-chemical properties of laser-ablated gold nanoparticles: In contrast to chemically synthesized gold nanoparticles, gold nanoparticles obtained by laser ablation do not contain any organic residues, in particular there is no need of stabilizing agents on the basis of citrate. Furthermore, the particles are free of gold-thiol bonds, which are necessary in chemically synthesized particles in order to achieve a stable binding of the transfection agent and the nucleic acid molecules bound thereto. Since the laser-ablated gold nanoparticles are essentially free of any organic residues, the particles comprise a freely accessible gold surface which leads to a higher carrier capacity with respect to the transfection agent, on the one hand, and the nucleic acid molecules to be transferred, on the other hand. Therefore, an improved loading with transfection agent and nucleic acid molecules resulting in a higher transfection efficiency is achieved.
Overall, it was not foreseeable at all that the use of laser-ablated gold nanoparticles is linked with the aforementioned advantageous effects when used as conjugated gold nanoparticles for the delivery of a gene coding for a liver-specific and/or liver-expressed protein. In this context, reference is also made to the working examples which show that laser-ablated gold nanoparticles lead to superior properties in comparison to chemically synthesized gold nanoparticles.
The conjugated gold nanoparticles according to the present invention are a promising candidate for the use in a therapeutic concept for the treatment of a variety of monogenetic disorders in order to introduce an intact copy of the mutated and/or deficient gene into the target cells. In this context, the conjugated gold nanoparticles are suitable for the transfection of liver cells.
In particular, the conjugated gold nanoparticles according to the present invention are suitable for the treatment of monogenetic disorders, particularly but not exclusively associated with an impaired and/or reduced hemostasis and/or blood clotting, especially wherein the disorder is a hemophilia, in particular hemophilia A and/or hemophilia B. Furthermore, the conjugated gold nanoparticles according to the present invention are suitable for the treatment of monogenetic lipometabolic disorders.
In other words, the conjugated gold nanoparticles according to the present invention are suitable to provide a long-term expression of the liver-specific and/or liver-expressed protein in the target cells, in particular liver cells. On this basis, it is possible to achieve an excellent efficacy of a therapeutic concept on the basis of the conjugated gold nanoparticles according to the present invention. When compared to genetic approaches known in the prior art as well as delivery systems on the basis of chemically synthesized gold nanoparticles, the concept according to the present invention is not only linked with an improved efficacy, but also with an improved safety, a lowered toxicity and a reduced number of required treatment units due to the highly efficient long-term expression of the liver-specific and/or liver-expressed protein.
Prior to further specifications of particularly preferred embodiments of the present invention, relevant definitions of terms used according to the present invention are given with respect to a better understanding of the claimed subject-matter:
The term “monogenetic disorder”, “monogenetic disease” or “single-gene disorder” refers to diseases or disorders, which result from modifications, in particular mutations, in a single gene occurring in all cells of the preferably human body. The mutations are in general linked with a partial or complete loss of the physiological function of the protein (“loss-of-function-mutation”). In particular, monogenetic disorders can result from sex-linked, recessive or dominant heredity. Furthermore, monogenetic disorders can result from sporadic mutations in a single gene.
According to the present invention, the term “nanoparticle” refers to particles having an average particle diameter between 1 and 100 nm. Nanoparticles according to the present invention are based on inorganic material, preferably ligand-free gold. Nanoparticles of this kind are particularly suitable for medical purposes, especially for the transfer and/or delivery of nucleic acid molecules, since they are substantially chemically inert. Surprisingly, on the basis of the present invention, gold nanoparticles have turned out as particularly well-suited carriers for nucleic acid molecules comprising nucleic acid sequences coding for liver-specific and/or liver-expressed proteins due to their non-toxicity and excellent biocompatibility, on the one hand, and their transfection efficiency, in particular with respect to liver cells, on the other hand. Gold nanoparticles are well tolerated in various mammals. After intravenous injection, they are preferably taken up by the liver and then excreted again via the bile.
“Laser ablation” in the sense of the present invention indicates a process of removing material from a solid surface, in particular gold, by irradiating the solid with a laser beam. According to a preferred embodiment of the present invention, removing of the material is performed with a pulsed laser, preferably by pulsed laser ablation in liquid (PLAL). The principal of pulsed laser ablation in liquid is based on focusing a laser beam on a solid target for ablation, in particular gold. The properties of the resulting particles, in particular the size, are controlled by the laser parameters used as well as solvent, temperature, pressure or wave length, pulse duration, energy or reputation rate. In general, the skilled practitioner is able to adapt the settings of the laser ablation to produce gold nanoparticles with the desired properties, in particular an appropriate size/diameter.
The term “polyethylenimine”, synonymous also “PEI”, “poly[imino(1,2-ethanediyl)]” a “polyaziridine”, as used according to the present invention, especially refers to a polycationic polymer with repeating units of an amine group and two carbon aliphatic CH2CH2 as a spacer between the repeating units of the amine groups. The chemical name of this polymer according to IUPAC is poly(iminoethylene). Linear polyethylenimines contain all secondary amines, wherein branched polyethylenimines contain primary, secondary and tertiary amino groups. Polyethylenimine was one of the first discovered transfection agents. When used as transfection agent—without being bound to this theory—, polyethylenimine condenses DNA into positively charged particles, which bind to anionic cell surface residues. The complex on the basis of DNA and polyethylenimine is then brought into the cell via endocytosis. Subsequently, the polyethylenimine causes an influx of water molecules into the endosomes, resulting in a bursting of the endosomes and a release of the DNA into the cytoplasm. According to the present invention, it was surprisingly found that polyethylenimines are not only suitable for the mediation of transfection as such, but also as a ligand for gold nanoparticles in order to build a gold nanoparticle/PEI/DNA complex. With respect to further information concerning polyethylenimine, reference is made to the encyclopedia RÖMPP Chemielexikon, 1999, 10th edition, Georg Thieme Verlag Stuttgart, New York, page 3448, key word “polyethylenimine”.
Examples for variants of polyethylenimine for the delivery system according to the present invention are commercially available from Sigma-Aldrich Chemie GmbH, Munich, DE (branched PEI, 25 kDa), Polysciences Inc., Warrington, US (linear PEI, 10 kDa; linear PEI, 25 kDa; linear PEI, commercially available under the tradename Transporter5™) and/or Polyplus Inc., Illkirch, FR (JetPEI™, linear PEI, JetPEI™-Hepatocyte, galactose-conjugated linear PEI).
The term “vector” is used for a DNA molecule which is suitable for the use as a vehicle to artificially carry foreign genetic material, in particular genetic material comprising a nucleic acid sequence coding for a liver-specific and/or liver-expressed protein and/or preferably physiologically active domains and/or fragments thereof, into target cells. According to a preferred embodiment of the present invention, the vector used in the conjugated gold nanoparticles is a non-viral or mini circle vector in order to improve the safety and compatibility when used in gene therapy. Particularly, the vector used according to the present invention does not integrate into the genome. Thereby, the vector used according to the present invention still provides for a efficient transfection of the target cells and allows for a long-term expression of the coding sequence, preferably on the basis of an episomal attachment to the chromosomal DNA. In contrast to known approaches with respect to gene therapy for the treatment of monogenetic disorders, the vector used according to the present invention is not a viral vector, in particular no vector on the basis of the adeno-associated virus (AAV).
The term “promoter” as used according to the present invention relates to a DNA (desoxyribonucleic acid) or nucleic acid sequence, in particular a regulatory sequence, which is required for the expression of a coding sequence linked to the promoter, in particular a corresponding coding sequence located 3′ or downstream to the promoter. In order to achieve a stable and reliable expression of the nucleic acid sequence coding for a liver-specific and/or liver-expressed protein, the nucleic acid molecules, in particular the vector, comprise preferably a promoter derived from a eukaryotic, in particular human gene or a promoter derived from a virus. On this basis, the compliance of the conjugated gold nanoparticles with the nucleic acid molecules, in particular the vector, on the one hand, in the patient and the efficiency of expression of the coding sequence, on the other hand, can be improved. A promoter according to the present invention can comprise a core promoter, including a transcription start site, a binding site for RNA polymerases and binding sites for general transcription factors.
The term “coding sequence”, “coding region” or “nucleic acid coding sequence” refers to a nucleic acid sequence coding for a protein or domains or fragments of a protein. Furthermore, the coding sequence can refer to a nucleic acid sequence coding for fusion proteins, in particular fusion proteins on the basis of a liver-specific and/or liver-expressed protein and an albumin. In other words, the coding sequence according to the present invention contains a nucleic acid sequence coding for a liver-specific and/or liver-expressed protein and/or domains and/or fragments thereof and can contain further nucleic acid sequences, which results in a coding sequence coding for a fusion protein. In particular, according to a preferred embodiment of the present invention, the coding sequence is based on the cDNA sequence coding for a protein and/or domains or fragments of a protein.
In the following, particularly preferred embodiments of the present invention are delineated:
According to the present invention it is preferred when the laser-ablated gold nanoparticles are obtained by pulse laser ablation in liquid (PLAL). The use of laser-ablated gold nanoparticles in the conjugated gold nanoparticles according to the present invention is linked with several advantages, in particular with respect to the efficacy of gene transfer and a safe application without undesired side effects or adverse reactions, as delineated before.
With respect to the production of the laser-ablated gold nanoparticles as such, i.e. the particles before conjugation or “naked” particles, it is particularly preferred to use pulsed laser irradiation with a wave length in the range from 3.300 to 1.500 nm, preferably in the range from 800 to 1.200 nm. On this basis, particles with a suitable size and an even particle size distribution are obtained. With respect to further information regarding the laser ablation, reference can also be made to the third aspect of the present invention, which relates to the method for the preparation of conjugated gold nanoparticles.
Furthermore, in this context it is preferred when the gold nanoparticles before conjugation, i.e. the naked particles, have an average particle diameter dp [nm] in the range from 0.01 to 100 nm, in particular 0.05 to 80 nm, preferably 0.1 to 50 nm, particularly preferred 0.5 to 30 nm, even more preferred 1 to 15 nm, especially preferred 2 to 10 nm, preferably determined by analytical disc centrifugation (ADC) and/or transmission electron microscopy (TEM) and/or UV/VIS spectra.
Particularly reliable results with respect to the determination of the particle size are obtained by analytical disc centrifugation. Analytical disc centrifuge is an analytical device that can accurately determine the size distribution of colloidal systems. The method is particularly suitable for microscopic to submicroscopic spherical particles with sizes between 3 nm and 100 μm. The analysis of the particles is based on the sedimentation principle, in which a separation by different radii of the particles takes place upon penetration of a liquid medium. Regarding particles of the same density, the larger particles sediment faster than the smaller particles. If spherical bodies are used, the sedimentation rate can be determined by the Stokes equation.
Further information with respect to the determination of the particle diameter of the gold nanoparticles on the basis of analytical disc centrifugation and/or transmission electron microscopy are evident from the scientific publication of Fissan et al.: “Comparison of different characterization methods for nanoparticle dispersions before and after aerosolization”, published in Anal. Methods, 2014, 6: 7324-7334, the disclosure of which is hereby incorporated by reference. With respect to the determination of the particle diameter of the gold nanoparticles by UV/VIS spectra, further information are evident from the scientific publication of Haiss et al.: “Determination of Size and Concentration of Gold Nanoparticles from UV-Vis Spectra”, published in Anal. Chem., 2007, 79(11), 4215-4221, wherein the disclosure of the publication, in particular with respect to the details of the determination methods, is hereby incorporated by reference.
The particle size is adjusted by variation of laser energy, wavelength of the pulsed laser irradiation and time. The adjustment of the particle as such is performed on the basis von general knowledge of the skilled practitioner. Preparation of the gold nanoparticles by pulsed laser ablation in liquid can be performed by using a picosecond laser (commercially available from Ekspla, Vilnius, Lithuania).
In particular, the uptake of the gold nanoparticles by the cells can be significantly increased on the basis of the use of gold nanoparticles having the aforementioned size. Furthermore, a purposeful selection of the size and/or average particle diameter is relevant with respect to avoid the potential toxicity of gold nanoparticles. In particular, gold nanoparticles with a size below the aforementioned ranges behave different in cells leading to a certain toxicity. Gold nanoparticles having a size above the aforementioned ranges, however, are not able to penetrate the cell membrane and are therefore not suitable for a transfer of nucleic acid molecules. The use of gold nanoparticles having the aforementioned sizes leads to an efficiency enhancement with respect to the transfection efficiency, on the one hand, and a reduced, preferably non-existent toxicity—in other words an improved biocompatibility—with respect to the cells.
With respect to the “naked”, i.e. the unconjugated gold nanoparticles, it is advantageous when the gold nanoparticles before conjugation have a gold surface, wherein at least 90%, preferably at least 95% of said gold surface is freely accessible and not attached to any molecules. On this basis, the loading capacity of the gold nanoparticles with nucleic acid molecules and transfection agent is improved. As a result, the transfection efficiency as such as well as the DNA transfer, in particular the endosomal release of the nucleic acid molecules after uptake by the cell, are further improved.
Furthermore, with respect to the particle size of the conjugated particles, it is advantageous when the conjugated gold nanoparticles have an average hydrodynamic diameter dhd [nm] in the range from 0.05 to 150 nm, in particular 0.1 to 100 nm, preferably 0.5 to 80 nm, particularly preferred 1 to 50 nm, even more preferred 2 to 40 nm, especially preferred 10 to 30 nm, preferably determined by the method of dynamic light-scattering.
With respect to the determination of the average hydrodynamic diameter of the conjugated nanoparticles, known methods for the measurement of the hydrodynamic diameter of nanoparticles are used, in particular dynamic light scattering. With respect to the determination of the hydrodynamic radius of the conjugated gold nanoparticles by dynamic light scattering, reference can be made to the publication according to Menendez-Manjon and Barcikowski: “Hyrodynamic size distribution of gold nanoparticles controlled by repetition rate during pulsed laser ablation in water”, published in Appl. Surf. Sci., Vol. 257, Issue 9, 2011.
In order to provide an efficient transfection, on the one hand, and a stable binding of the nucleic acid molecules, in particular the vector, on the other hand, the polyethylenimine and/or derivatives and/or salts thereof are bound to the gold nanoparticles, preferably through electrostatic interaction with the surface of the gold nanoparticles. In particular and without being bond to this theory, it is assumed that the electrostatic interaction is based on partial charges of the nitrogen atoms of the polyethylenimine, on the one hand, and the gold nanoparticles, on the other hand. The respective single electrostatic bonds are rather weak, but in total, i.e. the sum of all bonds, a bonding of the polyethylenimine to the gold nanoparticles is achieved, which is strong enough in order to provide stable conjugated gold nanoparticles but thereby still allowing an endosomal release of the nucleic acid molecules, in particular of the vector, after uptake by the cell.
With respect to the transfection agent, it is particularly preferred when the polyethylenimine and/or derivatives and/or salts thereof are selected from the group of (i) linear polyethylenimines and/or derivatives and/or salts thereof; (ii) branched polyethylenimines and/or derivatives and/or salts thereof; and/or (iii) monosaccharide-conjugated, preferably galactose-conjugated polyethylenimines and/or derivatives and/or salts thereof.
Polyethylenimine is a particularly efficient transfection agent with respect to the conjugated gold nanoparticles according to the present invention since it is highly compatible and linked with a high loading capacity with respect to the nucleic acid molecules, resulting in an efficient DNA transfer. Particularly good results with respect to compatibility and non-toxicity and furthermore with respect to transfection efficiency can be achieved with the use of linear polyethylenimines. Furthermore, the use of a monosaccharide-conjugated polyethylenimine, preferably galactose-conjugated polyethylenimine, is linked with an additional function of the polyethylenimine. For, on this basis a targeting of the conjugated gold nanoparticles is possible. In particular liver cells comprise in their membrane galactose specific cell surface receptors, for example galactose-specific membrane lectins as asialoglycoprotein receptors (ASGPR). By the use of a polyethylenimine conjugated with galactose, the conjugated gold nanoparticles can specifically bind to the respective receptors in the cell surface of liver cells, followed by an uptake of the conjugated gold nanoparticles by the cells. On this basis, the specificity of the conjugated gold nanoparticles according to the present invention can be further improved. Galactose conjugated polyethylenimine is commercially available from Polyplus Inc., Illkirch, FR under the tradename “JetPEI®-hepatocyte”.
Furthermore, according to a particularly preferred embodiment of the present invention, the conjugated gold nanoparticles comprise at least two layers of polyethylenimine and/or derivatives and/or salts thereof. With respect to this embodiment, it is particularly preferred when the conjugated gold nanoparticles comprise the at least two layers of polyethylenimine in the sense of a layer-by-layer assembly, i.e. an inner layer on the basis of polyethylenimine, wherein nucleic acid molecules are bound to this inner layer of polyethylenimine. The gold nanoparticles conjugated with said inner layer and nucleic acid molecules bound thereto are further conjugated with a second polyethylenimine layer and/or an outer layer on the basis of polyethylenimine.
In particular, the conjugated gold nanoparticles comprise alternating layers of polyethylenimine and/or derivatives and/or salts thereof and nucleic acid molecules, in particular an inner and an outer layer comprising polyethylenimine and/or derivatives and/or salts thereof, wherein nucleic acid molecules are bound to the inner and/or the outer layer.
With respect to the embodiment of the conjugated gold nanoparticles according to the present invention comprising of a layer-by-layer assembly, it is particularly intended that the inner layer comprises linear and/or branched, preferably linear polyethylenimines and/or derivatives and/or salts thereof. On this basis, the surface of the gold nanoparticles to be conjugated is covered with a sufficient amount of transfection agent providing a good loadability of the particles with nucleic acid molecules. After loading and/or conjugating the inner layer with nucleic acid molecules, the conjugated gold nanoparticles are covered or coated with an outer layer, also on the basis of polyethylenimines and/or derivatives and/or salts thereof. In this context, it is particularly preferred when the outer layer comprises linear, branched and/or monosaccharide-conjugated, preferably monosaccharide-conjugated polyethylenimines and/or derivatives and/or salts thereof.
On the basis of an outer layer and/or a layer-by-layer assembly of the polyethylenimine and the nucleic acid molecules, the transfection efficiency and the transfer of nucleic acid molecules is further improved. Furthermore, on the basis of the use of monosaccharide-conjugated polyethylenimines, in particular galactose-conjugated polyethylenimines, in the outer layer a specific targeting of the gold nanoparticles to liver cells is achieved.
Furthermore, the transfection efficiency and compatibility of the delivery system according to the present invention can be further improved on the basis of the use of polyethylenimines and/or derivatives and/or salts thereof having a defined number average molecular weight. In particular, it is preferred when the polyethylenimine and/or derivatives and/or salts thereof have a number average molecular weight Mn in the range from 10 Da to 200 kDa, in particular from 100 kDa to 150 kDa, especially from 1 kDa to 100 kDa, particularly from 2 kDa to 50 kDa, preferably from 5 kDa to 40 kDa, more preferably from 8 kDa to 30 kDa, for example determined by means of gel permeation chromatography and/or according to DIN 55672-3:2016-03. In this context, reference is made to the working examples performed by the applicant, which show that the purposeful selection of polyethylenimine and/or derivatives and/or salts thereof having a certain molecular weight leads to an improved transfection efficiency as well as a reduced toxicity.
With respect to a particularly compatible therapeutic concept with a lowered risk of undesired side effects, in particular an undesired integration of the nucleic acid molecules into the gene known, it is preferred that the vector is a non-viral and not integrating vector. In other words, the conjugated gold nanoparticles according to the present invention are designed for a non-viral approach with respect to transfection and gene delivery. In particular, the conjugated gold nanoparticles are free from vectors on the basis of adeno-associated viruses (AAV), lentiviruses, retroviruses, adenoviruses and hybrids on the basis of the aforementioned vector systems. In particular, this means that the transfection mechanisms used according to the present invention is not based on viral systems. It is still possible though that the vectors used in the conjugated gold-nanoparticles comprise promoter sequences or elements of viral origin for the regulation of transcription.
In order to provide a stable and/or specific expression of the coding sequence contained in the nucleic acid molecule, it is preferred when the promoter is inducible and/or constitutive in mammalian cells, in particular human cells, preferably liver cells and/or fibroblasts. Likewise, it is possible that the promoter directs a tissue-specific, in particular liver-specific expression of the coding sequence. On this basis, the specificity of the promoter or the specificity of the expression directed by the promoter is variable and can be purposefully tailored or adjusted. In particular, any promoter directing a preferably constitutive expression of the coding sequence in several mammalian cells, cell types or tissues can be used in the nucleic acid molecules in the conjugated gold nanoparticles according to the present invention. Likewise, on the basis of tissue-specific promoters, in particular liver-specific promoters, the expression of the coding sequence can be purposefully targeted or adjusted. In this context, the promoter can be tailored and/or selected depending on the target cells, the severeness of the monogenetic disorder and the coding sequence to be expressed.
According to the present invention, the specificity of the promoter or the specificity of the expression directed by the promoter is variable and can be purposefully tailored or adjusted. In particular, any promoter directing a preferably constitutive expression of the coding sequence in several mammalian cells, cell types or tissues can be used in the nucleic acid molecules, in particular the vectors. In particular, in connection with the expression of coding sequences having nucleic acid sequence coding for a protein involved in hemostasis, the use of a constitutively active promoter is preferred.
According to a preferred embodiment of the present invention, the promoter is derived from the gene coding for human Elongation Factor-1 alpha (EF1a). In particular, according to a further preferred embodiment of the present invention, the promoter is derived from the promoter of the gene coding for human Elongation Factor-1 alpha (EF1a) and the first intron and/or a fragment of the first intron of the gene coding for human Elongation Factor-1 alpha (EF1a). A promoter derived from human Elongation Factor-1 alpha (EF1a) directs a reliable and constant expression of the coding sequences in mammalian cells, in particular human cells, preferably liver cells and/or fibroblasts, especially hepatocytes and/or fibroblasts. In this context, reference is also made to the working examples performed by the applicant. The working examples performed by applicant show that different promoters derived from the gene coding for human Elongation Factor-1 lead to a stable long-term expression of the coding sequence in several cell types, for example liver cells or fibroblasts (cf. also working examples).
Furthermore, according to another preferred embodiment of the present invention, the promoter is derived from the human SERPINA1 promoter. The SERPINA1 promoter directs a reliable and constant expression of the coding sequences in mammalian cells, in particular human cells, preferably liver cells and/or fibroblasts.
According to another, likewise preferred embodiment of the present invention, the promoter is derived from the hAAT (human alpha1-antitrypsin) promoter. The use of this promoter is particularly suitable with respect to directing a constant and stable expression of the coding sequence in mammalian cells, in particular human cells, preferably liver cells and/or fibroblasts. In this context, reference can also be made to the working examples performed by the applicant. On the basis of the working examples, it can be seen that the hAAT promoter leads to a stable long-term expression of the coding sequence in various cell types, in particular liver cells or fibroblasts.
According to another preferred embodiment of the present invention, the promoter is derived from Cytomegalovirus (CMV), in particular human CMV. In other words, according to this embodiment of the present invention, the promoter is the CMV promoter. The CMV promoter directs a stable and reliable gene expression in several mammalian cell types, for examples liver cells, in particular hepatocytes, or fibroblasts. With respect to the expression level of the coding sequence, reference is made to the working examples performed by applicant, which verify the stable expression of the coding sequence under control of the CMV promoter.
Furthermore, according to the present invention it can be intended that the promoter comprises a codon-optimized nucleic acid sequence and/or a nucleic acid sequence optimized for human gene expression and/or human codon usage. In particular, this applies for embodiments with a promoter containing further regulatory elements, for example on the basis of introns or parts of introns of a gene, especially of the gene the promoter is derived from.
According to a preferred embodiment of the present invention, the promoter has a nucleotide sequence according to SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and/or SEQ ID NO. 5, preferably SEQ ID NO. 2, SEQ ID NO. 3 and/or SEQ ID NO. 4. Likewise, according to a preferred embodiment of the present invention, the promoter has a nucleic acid sequence having at least 85%, in particular at least 90%, preferably at least 95% identity with SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and/or SEQ ID NO. 5, preferably SEQ ID NO. 2, SEQ ID NO. 3 and/or SEQ ID NO. 4.
Particularly preferred promoter sequence contained in the nucleic acid molecules used in the conjugated gold nanoparticles according to the present invention is derived from the gene, in particular the promoter, of human Elongation Factor-1 alpha (EF1a). According to a preferred embodiment of the present invention, the promoter on the basis of EF1a contains a sequence optimized first intron, which has been considerably shortened. Furthermore, a cryptic splice side contained in the native nucleotide sequence has been deleted. The promoter according to SEQ ID NO. 2 and/or SEQ ID NO. 3 leads to a stable and highly efficient expression of the coding sequence in mammalian cells.
Likewise, according to another particularly preferred embodiment of the present invention, the nucleic acid molecules comprise the hAAT promoter in order to direct the expression of the coding sequence. In this context, it is preferred when the promoter has a nucleic acid sequence according to SEQ ID NO. 4.
In order to further enhance the expression of the coding sequence, it can be intended that the nucleic acid molecules, in particular the vector, contain at least one further cis-regulatory element, especially at least one further transcriptional enhancer.
According to a preferred embodiment of the present invention, the cis-regulatory element is derived from the apolipoprotein E gene, in particular the apolipoprotein E hepatic locus control region. The additional use of a cis-regulatory element on the basis of the apolipoprotein E hepatic locus control region (HCR) leads to an improved expression of the coding sequence in the target cells.
In this context, it is particularly preferred when the cis-regulatory element has a nucleotide sequence according to SEQ ID NO. 6 and/or when the cis-regulatory element has a nucleic acid sequence having at least 85%, in particular at least 90%, preferably at least 95% identity with SEQ ID NO. 6.
In particular, a further cis-regulatory element has been proven to be advantageous with respect to the expression efficiency when used together with a SERPINA1 promoter or a hAAT promoter.
A preferred design of the coding sequence contained in the nucleic acid molecules, in particular the vector, according to the present invention is delineated in the following:
In order to achieve an improved expression of the coding sequence, according to the present invention it is intended that the nucleic acid sequence of the coding sequence is codon-optimized for human gene expression and/or human codon usage. The introduction of synonymous mutations, i.e. mutations that lead to the same translational product, leads to an efficient enhancement of the protein expression. On the basis of a replacement of rare codons with preferred codons, the expression of the coding sequence and the production of the target protein in the target cells can be further improved.
With respect to the selection of the coding sequence, according to a preferred embodiment of the present invention, the coding sequence comprises a nucleic acid sequence coding for a liver-specific and/or liver-expressed protein selected from proteins produced and/or predominantly expressed in the liver. As delineated before, the production and secretion of proteins belong to the main functions of the liver. The proteins produced and secreted by the liver in particular include proteins involved in hemostasis, i.e. proteins regulating blood clotting. Mutations in genes coding for liver-specific and/or liver-expressed proteins can lead to a reduced or completely lacking production of the protein. Furthermore, mutations can result in the production of defective proteins, i.e. proteins that lost their physiological functionality (so called lost-of-function-mutation).
Factors involved in hemostasis and fibrinolysis are of particular importance for the present invention, since mutations in genes coding for such factors or proteins, in particular factors of the coagulation cascade, lead to a group of monogenetic disorders subsumed as hemophilia. Liver-specific and/or liver-expressed proteins involved in hemostasis and fibrinolysis are in particular all factors of the coagulation cascade, especially fibrinogen (FI), prothrombin (FII), tissue factor or tissue thromboplastin (FIII), proaccelurin or labile factor (FV), stable factor or proconvertin (FVII), antihemophilic factor A (FVIII), antihemophilic factor B, synonymously also known as Christmas factor (FIX), Stuart-Prower factor (FX), plasma thromboplastin antecedent (FXI), Hageman factor (FXII), fibrin-stabilizing factor (FXIII), von Willebrand factor (VWF), Fletcher factor, synonymous also prekallicrein, high-molecular weight kininogen or Fitzgerald factor, fibronectin, antithrombin III, heparin-co-factor II, protein-C, protein-S, protein-Z, plasminogen, alpha2-antiplasmin, tissue plasminogen activator, urokinase and plasminogen activator inhibitor-1 (PAI1). Mutations in genes coding for the aforementioned coagulation factors and related substances can lead to genetic disorders, in particular to different types or subforms of hemophilia.
Further liver-specific and/or liver-expressed proteins of particular interest with respect to the present invention are proteins of the amino acid metabolism, in particular fumarylacetoacetate hydrolase, p-hydroxyphenylpyruvate hydroxylase and/or phenylalanine-4-hydroxylase, antiproteases, in particular alpha-1 antitrypsin, proteins of the bilirubin metabolism, in particular uridine diphospho-glucuronosyltransferase, proteins of the urea cycle, in particular arginase, argininosuccinate synthase and/or ornithine transcarbamylase, proteins of the carbohydrate metabolism, in particular alpha-glucan phosphorylase, amylo-1,6-glucosidase and/or glucose-6-phosphatase, proteins of the proteoglycan metabolism, in particular idursulfase, proteins of the sphingolipid metabolism, in particular glucocerebrosidase, and/or proteins involved in transport processes, in particular p-type ATPase, cystic fibrosis transmembrane regulator and/or low-density lipoprotein (LDL) receptor.
According to a preferred embodiment of the present invention, the coding sequence has a nucleic acid sequence coding for a human liver-specific and/or liver-expressed protein selected from the group of:
In particular, mutations in genes coding for coagulation factors are associated with genetic disorders, which are commonly summed up as hemophilia, in particular hemophilia A (factor FVIII deficiency), hemophilia B (factor FIX deficiency), von Willebrand disease (von Willebrand factor deficiency) and the rare factor deficiencies including deficiencies in factor FI, FII, FV, FVII, FX, FXI, FXII and/or FXIII. The conjugated gold nanoparticles with the nucleic acid molecules, in particular the vectors, can be used to transfer an intact copy of the genes coding for coagulation factors into the target cells, in particular liver cells. On this basis, the physiological deficiency with respect to respective coagulation factor can be balanced and/or improved through the stable expression of the coding sequence in the target cells, in particular liver cells.
It is especially preferred when the coding sequence has a nucleic acid sequence coding for a coagulation factor, in particular coagulation factor FVII, FVIII, FIX, FX, FXI, FXII, FXIII and/or preferably physiologically active domains and/or fragments thereof, preferably coagulation factor FVIII, FIX and/or preferably physiologically active domains and/or fragments thereof.
More particularly preferred is an embodiment of the present invention, wherein the coding sequence has a nucleic acid sequence coding for coagulation factor FVIII and/or preferably physiologically active domains and/or fragments thereof. In hemostasis, factor FVIII functions as cofactor for factor FIXa, which is necessary for the formation of factor FX. Mutations, in particular loss-of-function-mutations, in the gene coding for factor FVIII are linked with hemophilia A.
According to a particularly preferred embodiment of the present invention, the coding sequence has a nucleic acid sequence coding for coagulation factor FVIII with a deleted B-domain. The native FVIII protein has a total length of 2.351 amino acids with the so-called B-domain constituting of 911 amino acids. The B-domain is a highly glycosylated region of the protein but is not required for the physiological procoagulation activity of FVIII. On the basis of the deletion of the B-domain and the replacement of the B-domain by a short 14 amino acid linker, a fully functional fragment of FVIII can be provided which shows—due to the reduction of the length—an improved expression in the target cells.
According to a likewise preferred embodiment of the present invention, the coding sequence has a nucleic acid sequence coding for coagulation factor FIX and/or preferably physiologically active domains and/or fragments thereof. The physiological function of factor FIX is, together with Ca2+, membrane phospholipids and a factor FVIII cofactor, the formation of factor FX. Mutations, especially loss-of-function-mutations, in the gene coding for coagulation factor FIX result in hemophilia B. Conjugated gold nanoparticles comprising a nucleic acid sequence coding for coagulation factor FIX are therefore suitable for the use in a gene therapy for the treatment of hemophilia B in order to balance the loss of function caused by the mutation.
According to a further preferred embodiment of the present invention, the coding sequence has a nucleic acid sequence coding for a fusion protein on the basis of a coagulation factor and/or preferably physiologically active domains and/or fragments thereof, in particular coagulation factor FVIII and/or FIX, preferably coagulation factor FIX, and an albumin and/or domains and/or fragments thereof. On the basis of a fusion of coagulation factors to albumin, the pharmacokinetic properties of the coagulation factors can be significantly improved. In particular, coagulation factors on the basis of fusions with albumin comprise an extended half-life time. On this basis, the treatment intervals of the patients suffering from monogenetic disorders, in particular hemophilia, can be prolonged, i.e. a less frequent dosing is enabled.
Nevertheless, the list of coding sequences is not exhaustive, since the nucleic acid sequences coding for any liver-specific and/or liver-expressed protein associated with a monogenetic disorder can be integrated into the nucleic acid molecules used in the conjugated gold nanoparticles.
According to a preferred embodiment of the present invention, the coding sequence has a nucleotide sequence coding for coagulation factor FVIII and/or preferably physiologically active domains and/or fragments thereof. According to a particularly preferred embodiment of the present invention the coding sequence has a nucleotide sequence according to SEQ ID NO. 7 and/or SEQ ID NO. 8, preferably SEQ ID NO. 8, and/or wherein the coding sequence has a nucleotide sequence having at least 85%, in particular at least 90%, preferably at least 95% identity with SEQ ID NO. 7 and/or SEQ ID NO. 8, preferably SEQ ID NO. 8. Likewise, the coding sequence can have a nucleic acid sequence corresponding to the nucleic acid sequence of the native cDNA coding for human coagulation factor FVIII and/or the coding sequence can code for a protein having an amino acid sequence according to SEQ ID NO. 9 and/or an amino acid sequence having at least 85%, in particular at least 90%, preferably at least 95% identity with SEQ ID NO. 9.
According to a likewise preferred embodiment of the present invention, the coding sequence comprises a nucleic acid sequence coding for coagulation factor FIX and/or preferably physiologically active domains and/or fragments thereof. With respect to the nucleic acid molecules comprising a coding sequence for expressions of a protein, which carries out the physiologically functions of coagulation factor FIX, according to a preferred embodiment of the present invention the coding sequence has a nucleotide acid sequence according to SEQ ID NO. 10, SEQ ID NO. 11 and/or SEQ ID NO. 12 and/or a nucleotide sequence having at least 85%, in particular at least 90%, preferably at least 95% identity with SEQ ID NO. 10, SEQ ID NO. 11 and/or SEQ ID NO. 12. Likewise, the coding sequence can have a nucleotide sequence corresponding to the nucleotide sequence of the native cDNA coding for human coagulation factor FIX and/or wherein the coding sequence codes for a protein having an amino acid sequence according to SEQ ID NO. 13 and/or SEQ ID NO. 14 and/or an amino acid sequence having at least 85%, in particular at least 90%, preferably at least 95% identity with SEQ ID NO. 13 and/or SEQ ID NO. 14.
According to a further preferred embodiment of the present invention, the coding sequence has a nucleic acid sequence coding for a fusion protein on the basis of a coagulation factor and/or preferably physiologically active domains and/or fragments thereof, in particular coagulation factor FVIII and/or FIX, preferably coagulation factor FIX, and an albumin and/or domains an/or fragments thereof.
On the basis of a fusion of coagulation factors to albumin, the pharmacokinetic properties of the coagulation factors can be significantly improved. In particular, coagulation factors on the basis of fusions with albumin comprise an extended half life time. On this basis, the treatment intervals of the patience suffering from monogenetic disorders, in particular hemophilia, can be prolonged, i.e. a less frequent dosing leads to desired therapeutic effect.
In this context, according to a preferred embodiment of the present invention, the coding sequence has a nucleotide sequence according to SEQ ID NO. 15 and/or SEQ ID NO. 16 and/or a nucleotide sequence having at least 85%, in particular at least 90%, preferably at least 95% identity with SEQ ID NO. 15 and/or SEQ ID NO. 16. Likewise, the coding sequence can code for a protein having an amino acid sequence according to SEQ ID NO. 17 and/or SEQ ID NO. 18 and/or an amino acid sequence having at least 85%, in particular at least 90%, preferably at least 95% identity with SEQ ID NO. 17 and/or SEQ ID NO. 18.
Nevertheless, the list of coding sequences is not exhaustive, since the nucleic acid sequences coding for any liver-specific and/or liver-expressed protein associated with a monogenetic disorder can be integrated into the nucleic acid molecules, in particular the vectors, used according to the present invention.
As delineated before, the conjugated gold nanoparticles according to the present invention are designed in order to provide a non-viral genetic approach for the treatment of monogenetic disorders. In other words, the conjugated gold nanoparticles are designed in order to import an intact copy of a gene coding for a liver-specific and/or liver-expressed protein into the target cells, preferably liver cells or fibroblasts, in order to provide a therapeutically efficient expression of the protein. Since the nucleic acid molecules, in particular the vectors, contained in the conjugated gold nanoparticles do not integrate or insert into the genome, there is a possibility that the transfected cells lose the transferred nucleic acid molecules during the cell cycle. According to the present invention, it was found that the long-term expression of the coding sequence is improved with an increase of the episomal persistence of the nucleic acid molecule in the target cells. In this context it was surprisingly found that the episomal persistence is significantly improved when the vector comprises a scaffold/matrix attachment region, in particular a scaffold/matrix attachment region derived from the gene coding for human Interferon-beta (IFN-beta).
The term “scaffold/matrix attachment region”, also indicated as “S/MAR element” or “scaffold-attachment region” or “matrix-associated region”, refers to DNA sequences of eukaryotic chromosomes where the nuclear matrix attaches. Scaffold/matrix attachment regions of the eukaryotic DNA consist of about 70% T-rich regions and naturally mediate the structural organization of the chromatin within in the nucleus. In particular, the S/MAR elements constitute anchor points of the DNA for the chromatin scaffold and serve to organize the chromatin into structural domains. According to the present invention, it was surprisingly found that the use of the nucleotide sequence of a scaffold/matrix attachment region in the nucleic acid sequences, in particular the vectors, mediates the attachment of the transfected nucleic acid molecules to the nuclear matrix or the chromatin. On this basis, the non-integration of the nucleic acid molecules or the vector can be assured, thereby still allowing a stable expression of the coding sequence and a replication of the introduced nucleic acid molecule in particular during the S-phase of mitosis. The use of a scaffold/matrix attachment region increases the long-term episomal persistence of the nucleic acid molecules or the vector in the transfected target cells. Overall, the use of a nucleic acid sequence derived from a scaffold/matrix attachment region of a human gene is linked with a central advantage of the present invention, namely the prevention of an integration of the transferred transgenic nucleic acid molecules into the genomic DNA of the target cells. On this basis, the risk of further mutations, which can lead to the occurrence of malignant cells, can be significantly reduced.
In this context, it is particularly preferred when scaffold/matrix attachment region and for the SIMAR element has a nucleotide sequence according to SEQ ID NO. 19 and/or SEQ ID NO. 20, in particular SEQ ID NO. 20, and/or a nucleotide sequence having at least 85%, in particular at least 90%, preferably at least 95% identity with SEQ ID NO. 19 and/or SEQ ID NO. 20, in particular SEQ ID NO. 20. With respect to the assembly of the elements of the nucleic acid molecules, in particular the vector, it is preferred when the nucleic acid sequence derived from the scaffold/matrix attachment region of a eukaryotic gene is located 3′ to the promoter and/or the coding sequence.
The vector used according to the present invention can contain further elements advantageous or necessary for directing a stable expression of the coding sequence in the target cells. On the basis of the general knowledge, the skilled practitioner is able to select such further elements.
In particular, the vector can contain a transcription termination signal. The term “transcriptional termination signal” or “polyadenylation signal” as used according to the present invention refers to the section of a nucleic acid sequence that marks the end of a gene and/or a coding sequence during transcription. This sequence mediates the transcriptional termination by providing signals in the newly synthesized mRNA that trigger processes, which release the mRNA from the transcriptional complex. With respect to the present invention, the use of any transcriptional terminator suitable for the use in humans can be intended. The selection of a transcriptional termination signal and/or a polyadenylation signal does not represent a problem for the skilled practitioner.
Additionally, in order to optimally direct the expression of the coding sequence, the arrangement of the different elements of nucleic acid sequences within the nucleic acid molecules, in particular the vector, is of significance. In context with explanations concerning the assembly and/or arrangement of the nucleic acid sequence elements within the vector, the term “5′ to . . . ” is used synonymously to “upstream to . . . ”. Likewise, the term “3′ to . . . ” is used synonymously to “downstream to . . . ”. In other words, the terms upstream (“5′ to . . . ”) and downstream (“3′ to . . . ”) relate to the 5′ to 3′ direction in which RNA transcription takes place. In relation to double-stranded DNA, upstream is toward the 5′ end of the coding strand for the respective coding sequence and downstream is toward the 3′ end of the coding strand.
According to a preferred embodiment of the present invention, the promoter is located 5′ to the coding sequence and optionally the nucleic acid sequence derived from a scaffold/matrix attachment region of a human gene and/or a transcriptional termination signal. In particular, the elements, especially the promoter and the coding sequence, are arranged that the promoter can direct the expression of the coding sequence. Likewise, according to a preferred embodiment of the present invention, the optional nucleic acid sequence derived from the scaffold/matrix attachment region of a eukaryotic, in particular human gene is located 3′ to the promoter and/or the coding sequence. On this basis, a stable expression of the coding sequence and a high episomal persistence are provided.
With respect to a transcriptional termination signal in the vector, it is preferred when the transcriptional termination signal is located 3′ to the promoter and/or the coding sequence and/or optionally to a nucleic acid sequence derived from the scaffold/matrix attachment region of a human gene. As delineated before, the transcriptional termination signal is located such that the termination of the transcription of the coding sequence is enabled.
With respect to the transfection mediated by the conjugated gold nanoparticles according to the present invention it was found that transfection efficiency is not only influenced by gold nanoparticles, transfection reagent and nucleic acid molecules as such, but also by their proportions or ratios to one another, as delineated in the following:
In particular the transfer of nucleic acid molecules into the target cells can be improved on the basis of a defined weight related ratio of polyethylenimine to nucleic acid molecules. Particularly good results are achieved, when the weight related ratio of polyethylenimine to nucleic acid molecules is in the range of from 1:100 to 60:1, in particular from 1:50 to 40:1, especially from 1:30 to 20:1, preferably from 1:10 to 10:1, more preferred from 1:1 to 10:1, further preferred from 1:1 to 6:1. Likewise, it is preferred when the weight related ratio of polyethylenimine and/or derivatives and/or salts thereof to gold nanoparticles is in the range of from 1:100 to 100: 1, especially from 1:50 to 50:1, preferably from 1:30 to 20:1, in particular preferred from 1:20 to 10:1, even more preferred from 1:10 to 1:1.
With respect to the weight related ratios of the component of the delivery system according to the present invention, reference is also made to the working examples performed by applicant, which show that a purposefully selected weight related ratio leads to an improvement of the transfection efficiency and the resulting transfer of nucleic acid molecules into the target cells.
Overall, the conjugated gold nanoparticles according to the present invention are suitable for the use in the treatment, in particular a non-viral gene therapy, of a monogenetic disorder resulting from a mutation in a gene coding for a liver-specific and/or liver-expressed protein. In particular, the conjugated gold nanoparticles are able to transfer a intact copy of a gene coding for a liver specific and/or liver-expressed protein by transfection into the target cells, in particular mammalian cells, preferably human cells, for example liver cells of fibroblasts.
In context with the use of the gold nanoparticles according to the present invention it is particularly preferred when the monogenetic disorder is associated with an impaired and/or reduced hemostasis and/or blood clotting, especially wherein the disorder is a hemophilia, in particular hemophilia A and/or hemophilia B.
A further subject of the present invention is—according to a second aspect of the present invention—the use of conjugated gold nanoparticles as described before in the treatment, in particular a non-viral gene therapy, of a monogenetic disorder resulting from a mutation in a gene coding for a liver-specific and/or liver-expressed protein, and/or for the preparation of a medicament for the treatment, in particular a non-viral gene therapy, of a monogenetic disorder resulting from a mutation in a gene coding for a liver-specific and/or liver-expressed protein, preferably via transfection.
In this context, it is particularly preferred when the monogenetic disorder is associated with an impaired and/or reduced hemostasis and/or blood clotting, especially wherein the disorder is a hemophilia, in particular hemophilia A and/or hemophilia B.
For further details concerning this aspect of the invention, reference can be made to the above explanations in relation to the first inventive aspect, referring to the conjugated gold nanoparticles according to the present invention, said explanations also applying accordingly with regard to this aspect of the invention.
A further subject of the present invention is—according to a third aspect of the present invention—a method for the preparation of conjugated gold nanoparticles, wherein the gold nanoparticles comprise polyethylenimine (PEI) and/or derivatives and/or salts thereof, in particular conjugated gold nanoparticles as described before, and
wherein the method comprises the following method steps:
The method described in the following is particularly suitable in order to provide conjugated gold nanoparticles as described before according to the first aspect of the present invention.
Prior to further specifications of particularly preferred embodiments of the method according to the present invention, relevant definitions of terms are given with respect to a better understanding.
The term “unconjugated” or “naked” gold nanoparticle means that the surface of the gold nanoparticles is substantially free of any molecular attachments, in particular organic resins or side products. According to a preferred embodiment of the present invention the naked and/or unconjugated gold nanoparticles comprise a gold surface, wherein the gold surface is to at least 90%, preferably at least 95%, even more preferred to at least 99% not attached to any molecules and freely accessible. In other words, on the basis of laser ablation, in particular pulsed laser ablation in liquid, ligand-free gold nanoparticles are synthesized.
The laser ablation, in particular the pulsed laser ablation in liquid, is known to the skilled practitioner, as already delineated with regard to the conjugated gold nanoparticles as such. The following settings of the laser ablation has been proven to be particularly advantageous with respect to the properties of the gold nanoparticles against the background of an improved therapeutic concept for the treatment of monogenetic disorders.
According to a preferred embodiment of the present invention, laser ablation is performed with a pulsed laser irradiation having a wave length in the range from 330 to 1,500 nm, preferably in the range from 800 to 1,200 nm.
Furthermore, according to another preferred embodiment of the present invention, the pulse energy is in the range of 1 to 1,000 μJ especially 5 to 500 μJ, particularly 10 to 250 μJ, preferably 50 to 200 μJ, even more preferred 90 to 150 μJ.
With respect to the pulse repetition rate it is advantageous when the pulse repetition rate is in the range of 1 to 1,000 kHz, especially 5 to 500 kHz, particularly 10 to 250 kHz, preferably 50 to 200 kHz, even more preferred 80 to 150 kHz.
Furthermore, it is advantageous when the pulse duration is in the range of 0.1 to 500 ps, especially 0.5 to 100 ps, particularly 1 to 50 ps, preferably 2 to 25 ps, even more preferred 5 to 15 ps.
On the basis of the aforementioned parameters, gold nanoparticles are produced, which are particularly suitable for the use in the medical field, in particular a non-viral gene therapy. In this context, it is of particular interest, that the gold nanoparticles are produced with an average particle diameter that allows the gold nanoparticles to be taken up by cells, in particular mammalian cells, preferably human cell types. Nevertheless, the particle size should not be linked with a higher cell toxicity.
Therefore, according to a preferred embodiment of the present invention, the the gold nanoparticles are adjusted to an average particle diameter dp [nm] in the range from 0.01 to 100 nm, in particular 0.05 to 80 nm, preferably 0.1 to 60 nm, particularly preferred 0.5 to 50 nm, even more preferred 1 to 25 nm, especially preferred 2 to 10 nm, preferably determined by analytical disc centrifugation (ADC) and/or transmission electron microscopy (TEM) and/or UV/VIS spectra. As delineated in connection with the conjugated gold nanoparticles as such, are particles with the aforementioned sizes able to be taken up by the cell and thereby still non-toxic.
The particle size, in particular the average particle diameter, is adjusted by variation of laser energy, wavelength of the pulsed laser irradiation, pulse duration, repetition rate and duration of laser ablation. The above-described parameters are particularly suitable in order to provide particles having the preferred sizes, which enable the gold nanoparticles to cross the membrane of the target cells without showing a significant toxicity or immunogenicity.
According to a preferred embodiment of the present invention, a gold target is used for laser ablation, wherein the gold nanoparticles are ablated from such gold target. In this context, it is particularly preferred when the gold target has a thickness in the range of 0.1 to 20,000 μm, especially 1 to 15,000 μm, particularly 10 to 10,000 μm, preferably 50 to 8,000 μm, even more preferred 100 to 5,000 μm. It is particularly preferred to use gold foil as gold target for laser ablation.
In order to provide a good compatibility of the gold nanoparticles, it is preferred when laser ablation is performed in a non-toxic, compatible liquid and/or medium. Therefore, according to a preferred embodiment of the present invention, laser ablation, in particular pulsed laser ablation in liquid, is performed in (i) purified water and/or (ii) phosphate based buffer, preferably sodium phosphate buffer (NaPB) and/or phosphate buffer saline (PBS) as liquid.
The conjugation of the gold nanoparticles with polyethylenimine as transfection reagent, i.e. method step (b) according to the present invention, can be performed in different ways, which are delineated in the following:
With respect to a first preferred embodiment of the present invention, method step (b) and/or conjugating the gold nanoparticles with polyethylenimine and/or derivatives and/or salts thereof is performed simultaneously with method step (a) and/or laser ablation of the unconjugated (naked) gold nanoparticles. In this context, the laser ablation, in particular the pulsed laser ablation in liquid, is performed in the presence of polyethylenimine and/or derivatives and/or salts thereof.
According to this preferred embodiment it was surprisingly found that a stable conjugation of the gold nanoparticles with polyethylenimine can be achieved on the basis of the addition of the transfection agent to the liquid used for laser ablation. In this context, it was particularly surprising that the laser pulses are not hindering or interfering with respect to the interaction between the gold nanoparticles and the transfection agent. For, the bonding of the transfection reagent to the gold nanoparticles is based on rather weak electrostatic interactions on the basis—without being bound to this theory—of the partial charges of gold, on the one hand, and the nitrogen atoms of the transfection agent, on the other hand. Despite the high energy input by the laser, a sufficient conjugation of the gold nanoparticles with the transfection reagent is achieved according to this embodiment of the method.
In order to achieve a good loadability of the conjugated gold nanoparticles with nucleic acid molecules and to provide an efficient gene transfer and uptake of the particles by the cells, it is particularly preferred when polyethylenimine and/or derivatives and/or salts thereof is added to the liquid, especially wherein polyethylenimine and/or derivatives and/or salts thereof is added to a concentration in the range from 0.1 to 1.000 μg/ml, especially in the range from 0.5 to 800 μg/ml, preferably in the range from 5 to 500 μg/ml, in particular in the range from 10 to 300 μg/ml, particularly preferred in the range from 20 to 200 μg/ml, based on the liquid for pulsed laser ablation.
According to a second, likewise preferred embodiment of the present invention, conjugating the gold nanoparticles with the transfection agent polyethylenimine is performed after generating the unconjugated, naked gold nanoparticles by laser ablation:
According to this further preferred embodiment of the present invention, method step (b) and/or conjugating the gold nanoparticles with polyethylenimine and/or derivatives and/or salts thereof is performed by admixing the laser-ablated gold nanoparticles with polyethylenimine and/or derivatives and/or salts thereof. In particular, according to this embodiment of the present invention admixing the gold nanoparticles with polyethylenimine and/or derivatives and/or salts thereof is performed as a separate method step and/or simultaneously with method step (c), i.e. the conjugation of the gold nanoparticles with nucleic acid molecules.
Both embodiments of the present invention with respect to conjugating the gold nanoparticles with the transfection reagent lead to highly competent conjugated gold nanoparticles with a high loadability for nucleic acid molecules and a high transfection efficiency, in particular an improved ability to cross the membrane of the target cells with subsequent endosomal release of the nucleic acid molecules.
With respect to method step (c) and/or conjugating the gold nanoparticles with nucleic acid molecules, admixing the nucleic acid molecules with the nanoparticles can be performed immediately before and/or within tranfection.
According to the present invention it was surprisingly found that gold nanoparticles obtained by laser ablation, in particular pulsed laser ablation in liquid, provide a particularly good loadability with respect to the transfection agent and the nucleic acid molecules.
Particularly good results with respect to the transfer of genetic material as well as the transfection efficiency are achieved when the conjugated gold nanoparticles prepared by the method of the present invention comprise the nanoparticles and the transfection agent in a defined weight related ratio. In order to provide such gold nanoparticles with improved properties, it is preferred when polyethylenimine and/or derivatives and/or salts thereof and gold nanoparticles are employed in the method of the present invention in a weight related ratio in the range from 1:100 to 100:1, especially from 1:50 to 50:1, preferably from 1:30 to 20:1, in particular preferred from 1:20 to 10:1, even more preferred from 1:10 to 1:1.
In this context, with respect to an efficient load of the conjugated gold nanoparticles with nucleic acid molecules, it is also preferred when polyethylenimine and/or derivatives and/or salts thereof and nucleic acid molecules are employed in a weight related ratio of polyethylenimine and/or derivatives and/or salts thereof to nucleic acid molecules in the range from 1:100 to 150:1, especially from 1:50 to 100:1, preferably from 1:20 to 50:1, in particular preferred from, 1:10 to 20:1, even more preferred from 1:1 to 10:1.
Overall, the high loadability of the laser-ablated gold nanoparticles with polyethylenimine, on the one hand, and nucleic acid molecules, on the other hand, was completely surprising and not foreseeable at all. Particularly good results with respect to transfection efficiency and gene transfer efficiency are achieved, when transfection agents, gold nanoparticles and nucleic acid molecules are employed in the above describe weight related ratios.
According to a particularly preferred embodiment of the present invention, the method for preparation of gold nanoparticles is suitable to provide conjugated gold nanoparticles comprising a so called layer-by-layer assembly on the basis of alternating layers of polyethylenimine and nucleic acid molecules. In order to provide such layer-by-layer assembly, subsequent to method steps (a) to (c) a method step further method step (d) is performed, wherein in method step (d) the particles obtained by method steps (a) to (c) are conjugated with a further outer layer comprising polyethylenimine and/or derivatives and/or salts thereof, preferably galactose-conjugated polyethylenimine and/or derivatives and/or salts thereof.
As described above in connection with the conjugated gold nanoparticles as such, a layer-by-layer assembly is advantageous with respect to an increase of the transfection efficiency. Furthermore, on the basis of galactose-conjugated polyethylenimine in the outer layer, conjugated gold nanoparticles allowing a purposeful targeting of the transfection of the target cells, in particular liver cells, can be prepared.
Overall, the present invention does not only provide conjugated gold nanoparticles as such, but also a method which is suitable to obtain such particles.
For further details concerning this aspect of the invention, reference can also be made to the above explanations with respect to the aspects outlined before, said explanations also applying accordingly with regard to the method according to the present invention.
Furthermore, subject-matter of the present invention—according to a fourth aspect of the present invention—is a nanoparticle-based delivery system for a coding sequence, preferably for the use in the treatment, in particular non-viral gene therapy, of a monogenetic disorder resulting from a mutation in a gene coding for a liver-specific and/or liver-expressed protein, wherein the delivery system comprises conjugated gold nanoparticles as described above according to the first aspect of the present invention and a physiologically and/or pharmaceutically acceptable carrier.
According to a preferred embodiment, the nanoparticle-based delivery system is prepared as a medicament, drug, pharmaceutical drug and/or agent, i.e. the nanoparticle-based delivery system is prepared as a drug used to diagnose, cure, treat or prevent diseases, in particular monogenetic disorders, as described before.
With respect to a particular preferred embodiment of the present invention, the nanoparticle-based delivery system is prepared for a systemic application, in particular an intravenous and/or oral, preferably systemic application.
With respect to the use of the nanoparticle-based delivery system according to the present invention it is preferred when the disorder or disease to be treated is associated with an impaired and/or reduced hemostasis and/or blood clotting, especially wherein the disorder is a hemophilia, in particular hemophilia A and/or hemophilia B.
For further information with respect to this aspect of the present invention, reference can also be made to the afore described aspects, wherein said explanations with respect to the aforementioned aspects also apply accordingly with respect to this aspect of the present invention.
Also subject-matter of the present invention is—according to a fifth aspect of the present invention—the use of a delivery system as described before in the treatment, in particular a non-viral gene therapy, of a monogenetic disorder resulting from a mutation in a gene coding for a liver-specific and/or liver-expressed protein and/or for the preparation of a medicament for the treatment of a monogenetic disorder resulting from a mutation in a gene coding for a liver-specific and/or liver-expressed protein.
In this context, it is particularly preferred when monogenetic disorder is associated with an impaired and/or reduced hemostasis and/or blood clotting, especially wherein the disorder is a hemophilia, in particular hemophilia A and/or hemophilia B.
For further details concerning this aspect of the present invention, reference can be made to the above explanations in relation to the aspects outlined before, said explanations also applying accordingly with regard to this aspect of the present invention.
Additionally, subject-matter of the present invention is—according to a sixth aspect of the present invention—a method for the transfection of target cells, especially mammalian cells, preferably human cells, preferably liver-cells and/or fibroblasts, wherein conjugated gold nanoparticles as described before are used in that method.
With respect to the method for transfection of target cells, reference is made to the above-described aspects of the present invention as well as the working examples.
For further details concerning this aspect of the present invention, reference can be made to the above explanations in relation to the aspects outlined before, said explanations also applying accordingly with regard to this aspect of the present invention.
Furthermore, subject-matter of the present invention is—according to a seventh aspect of the present invention—a transfected cell, preferably mammalian, in particular human cell, especially for the use in the treatment, in particular non-viral gene therapy, of a monogenetic disorder resulting from a mutation in a gene coding for a liver-specific and/or liver-expressed protein, wherein transfection has been performed with conjugated gold nanoparticles as described above and/or wherein the transfected cell comprises conjugated gold nanoparticles as described above.
With respect to the transfected cells, reference is made to the above-described aspects of the present invention as well as the working examples.
For further details concerning this aspect of the present invention, reference can be made to the above explanations in relation to the aspects outlined before, said explanations also applying accordingly with regard to this aspect of the present invention.
Finally, subject-matter of the present invention is—according to an eighth aspect of the present invention—a vector, in particular non-viral vector, preferably for the expression of a liver-specific and/or liver-expressed protein and/or preferably physiologically active domains and/or fragments thereof in a patient suffering from a monogenetic disorder caused by a mutation in the gene coding for the liver-specific and/or liver-expressed protein, wherein the vector comprises:
The vector according to the present invention is particularly suitable for the use in conjugated gold nanoparticles according to the present invention. In particular, the vector allows an expression of the coding sequence in the transfected target cells, preferably in order to compensate an impaired or total loss of the endogenous production of the respective liver-specific and/or liver-expressed protein.
With respect to the elements of the vector, in particular the coding sequence, the scaffold/matrix attachment region and the promoter, reference can be made to above explanations with respect to the nucleic acid sequences, in particular the vector, used in the conjugated gold nanoparticles according to the first aspect of the present invention.
However, according to a particularly preferred embodiment of the present invention, the promoter is derived from the gene coding to human Elongation Factor-1 alpha (EF1a) and/or from the human SERPINA1 promoter and/or from the hAAT (human 1-antitrypsin) promoter.
With respect to the promoter, it is particularly preferred when the promoter comprises a nucleotide sequence according to SEQ ID NO. 3, SEQ ID NO. 4 and/or SEQ ID NO. 5, especially SEQ ID NO. 3 and/or SEQ ID NO. 4. Likewise, the promoter can comprise a nucleic acid sequence having at least 85%, in particular at least 90%, preferably at least 95% identity with SEQ ID NO. 3, SEQ ID NO. 4 and/or SEQ ID NO. 5, especially SEQ ID NO. 3 and/or SEQ ID NO. 4.
Furthermore, it is also possible that the vector contains at least one further cis-regulatory element, especially at least one further transcriptional enhancer. In this context, it is particularly preferred when the cis-regulatory element is derived from the apolipoprotein E gene, in particular the apolipoprotein E hepatic locus control region (HCR). According to a preferred embodiment of the present invention, the cis-regulatory element has a nucleotide sequence according to SEQ ID NO. 6. Likewise, the cis-regulatory element can have a nucleic acid sequence having at least 85%, in particular at least 90%, preferably at least 95% identity with SEQ ID NO. 6.
For further details concerning this aspect of the present invention, reference can be made to the above explanations in relation to the aspects outlined before, said explanations also applying accordingly with regard to this aspect of the present invention.
Further advantages, properties and features of the present invention are apparent from the following description of preferred examples of the present invention shown in the drawings:
According to a preferred embodiment of the present invention, the conjugated gold nanoparticle 1 comprises a gold nanoparticle 2. The gold nanoparticle 2 comprises electrostatically bound polyethylenimine 3 and/or derivatives and/or salts thereof. In particular, the gold nanoparticle 2 is coated with polyethylenimine 3. Furthermore, on the basis of the polyethylenimine 3, nucleic acid molecules 4 are bound to the polyethylenimine/nanoparticle complex. On this basis, the polyethylenimine 4 fulfills several functions in the conjugated gold nanoparticles according to the present invention. On the one hand, the polyethylenimine 3 mediates the binding of the nucleic acid molecules 4 to the surface of the gold nanoparticles 2. On the other hand, polyethylenimine serves as the transfection reagent in order to improve the transfer of the nucleic acid molecules into the cells, in particular—without being bound to this theory—on the basis of the proton sponge effect, as delineated herein after in connection with
In this context, naked laser-ablated gold nanoparticles 2 are conjugated with a first polyethylenimine 3A, wherein this first polyethylenimine forms a first or inner layer on the surface of the gold nanoparticles. Subsequent to the first conjugation step, the polyethylenimine/gold nanoparticle complex is conjugated with nucleic acid molecules 4, which bind to the first polyethylenimine 3A. After adding the nucleic acid molecules 4, the conjugated gold nanoparticles 1, i.e. the polyethylenimine/gold nanoparticle/nucleic acid molecules complexes, are conjugated with a second polyethylenimine 3B and/or 3C. According to a preferred embodiment of the present invention, the second polyethylenimine 3C comprises a targeting unit, in particular on the basis of a conjugation with galactose. An outer layer on the basis of galactose-conjugated polyethylenimine allows a specific targeting of the conjugated gold nanoparticles to liver-cells, as delineated above. Likewise, the second polyethylenimine 3B can be identical to the polyethylenimine of the first and/or inner layer or any other of the above-mentioned polyethylenimines. Furthermore, the outer layer can also be based on a combination of a galactose-conjugated polyethylenimine and any other polyethylenimine used according to the present invention.
Starting point are conjugated gold nanoparticles according to the present invention, in particular as depicted in
The uptake of the conjugated gold nanoparticles into the cells occurs by endocytosis (B), resulting in the formation of an endosome 6 (C), which contains the conjugated gold nanoparticle 1 carrying the nucleic acid molecules 4 to be transferred. From the endosomes 6, the nucleic acid molecules 4 cannot directly enter the cytoplasm. On the basis of the polyethylenimine 3 bound to the gold nanoparticles 2, water molecules flow into the endosomes (D), causing the endosomes to burst (E). As a result, the nucleic acid molecules 4 to be transferred for transgenic expression of a coding sequence in the target cells are released into the cytoplasm (F).
The nuclear import (G) of the nucleic acid molecules 4 into the nucleus 9 then occurs passively during cell division after dissolution of the nuclear membrane or actively in non-dividing cells through nuclear pores 8 on the basis of transport molecules, in particular importins 7. In the nucleus 9, the nucleic acid molecules 4 bind to the core matrix and are replicated and expressed, resulting in the production of the liver-specific and/or liver-expressed protein.
With respect to a use in gene therapy, gold nanoparticles are mainly taken up by the liver after intravenous injection when used as carriers for nucleic acid sequences. Therefore, the conjugated gold nanoparticles according to the present invention comprise by nature a high specificity for the liver. According to the present invention, the binding of the conjugated nanoparticles to the surface of the liver cells is—without being bound to this theory—mediated by the transfection reagent on the basis of polyethylenimine. Since the conjugated gold nanoparticles according to the present invention as such already provide a high liver-specificity, a specific targeting is not necessarily needed in order to achieve a sufficient transfection of liver cells. Nevertheless, according to a particularly preferred embodiment of the present invention, galactose-conjugated polyethylenimine can be used for targeting.
The vectors as illustrated in
The vectors pEPI1-SM-L as shown in
The vector pEFi1-F9Pco as shown in
The vector peSEREG as shown in
The vector pcDNA3F9PwtInt1 as shown in
The vector pcDNA3F9Pco as shown in
The vector pcDNA3F9Pco_int1 according to
The vector pEFi43_F9Pco according to
The vector pEFi43F9Pwtint1 as shown in
The vector pEFi43F9PcoInt1 as shown in
The vector pEFi43F9PcoI2EG as shown in
The vector pEFi43F9PcoT2AEG as shown in
The vector peAATEG as shown in
According to this approach, 25 kDa linear PEI was used in two different amounts, i.e. 18 μg and 9 μg, and two different weight related ratios of polyethylenimine to nucleic acid molecules, i.e. 3:1 and 6:1 per well. For conjugation, gold nanoparticles with an average particle diameter of 5 nm (generated by pulsed laser ablation in liquid) and linear 25 kDa polyethylenimine (commercially available from Sigma-Aldrich/Merck KGaA, Darmstadt, DE) were pre-incubated the day before transfection and dialyzed against water with a 50 kDa molecular weight cut-off. Chemically synthesized gold nanoparticles with an average particle diameter of 5 nm and covalently bound 25 kDa linear PEI have been obtained from Nanopartz Inc., Loveland, Colo., US. For further conjugation of the gold nanoparticles with the vector DNA, the nucleic acid molecules have been added to the laser-ablated particles in an amount of 1.5 μg, 3 μg and 6 μg per well. Chemically synthesized gold nanoparticles were further conjugated with 350 μg, 1 μg, 3 μg, 6 μg or 20 μg nucleic acid molecules. The conjugated gold nanoparticles were incubated with 200,000 cells per well of a 6-well plate and GFP expression was analyzed three days after transfection by flow cytometry.
With respect to the preparation of the conjugated gold nanoparticles, gold foils have been used as gold target for the generation of gold nanoparticles with an average particle diameter of 5 nm by pulsed laser ablation in liquid (PLAL). PLAL has performed in solutions containing the above-mentioned concentrations of branched polyethylenimine. The different concentrations of the transfection agent were chosen to define optimal properties concerning the stability of the conjugated gold nanoparticles, gene transfer and toxicity effects. The gold nanoparticles conjugated or complexed with the transfection agent comprised after laser ablation an increased hydrodynamic diameter in the range of 14 to 22 nm, determined by dynamic light scattering. For the purpose of transfection, conjugated gold nanoparticles were prepared by adding 2 μg, 6 μg and 9 μg of nucleic acid molecules to 30 μg gold nanoparticles generated and complexed with the transfection reagent by pulsed laser ablation in liquid. The mixture was added to the cells, wherein each well of a 6-well plate contained 300,000 cells. After 4 hours and 24 hours, the cell culture medium was exchanged and cells were kept in culture for additional three days. Thereafter, HLF cells were collected and analyzed by flow cytometry.
In particular, a stable amount of gold nanoparticles of 30 μg, Transporter5™ and nucleic acid molecules (each 3 μg) were transfected with different amounts of the second transfection reagent (up to 9 μg). For this purpose, 3 μg of nucleic acid molecules were mixed with 30 μg laser-ablated gold nanoparticles having a size of 5 nm that were covered before with 9 μg transfection reagent on the basis of Transporter5™. Th After adding the nucleic acid molecules, a second layer of Transporter5™ or jetPEI®-hepatocyte was applied and added to the cells (300,000 cells/well in a 6-well format). Cell medium was exchanged 4 and 24 hours after transfection. Cells were kept in culture for two additional days and then analyzed by flow cytometry to determine the percentage of GFP expressing cells.
It can be seen from
The following working examples better illustrate the subject-matter of the present invention, and they should not be considered limiting the application.
In order to illustrate the present invention, in particular the underlying principles and advantages, various transfection studies have been performed, as delineated in the following.
Pulsed Laser Ablation in Liquid (PLAL)
The preparation of ligand-free (naked) gold nanoparticles has been performed with the method of pulsed laser ablation in liquid. For this purpose, a picosecond laser (available from Ekspla Atlantic, Vilnius, Lithuania) has been used. The laser ablation has been performed in phosphate buffered saline or sodium phosphate buffer (NaPP) as liquid with a pulse duration of 8 to 15 ps, up to 160 μJ pulse energy, a repetition rate of 80 to 150 kHz and a wavelength of 1,064 nm. Furthermore, the ablation was carried out in a 30 ml batch chamber for 10 min duration. As gold target, gold foil with a thickness of about 500 μm has been used.
Gold nanoparticles with a size of 10 nm or less have been obtained by using 600 μM sodium phosphate buffer (NaPP) as the liquid for laser ablation. In order to harvest particles with an average particle diameter of 10 nm or less, particles of larger size have been separated by ultracentrifugation (30,000×g, 17 min, 7° C.). While larger particles have been pelleted and discarded, particles of smaller size smaller than 10 nm remained in the supernatant and have been kept for further processing, i.e. conjugation with transfection reagent and nucleic acid molecules.
Larger particles with a size of 10 nm or more, in particular 40 to 60 nm, have been synthesized in purified water or phosphate buffered saline as liquid. The particles with a size of 10 nm or more, in particular 40 to 60 nm, have been obtained by incubating the particles after laser ablation for riping for at least 24 hours. After the incubation time the particles have been centrifuged (for example at 10,000 rpm, 70 min, 7° C.) to remove smaller particles. The supernatant containing smaller particles has been discarded while the larger particles in the pellet were re-suspended in suitable medium, for example purified water or a non-toxic buffer.
The unconjugated “naked” particles obtained by pulsed laser ablation in liquid have been conjugated with polyethylenimine and nucleic acid molecules, as delineated hereinafter.
Conjugation with polyethylenimine During Pulsed Laser Ablation in Liquid
According to a preferred embodiment of the method according to the present invention, conjugation of the gold nanoparticles with the transfection agent was performed simultaneously with pulsed laser ablation in liquid. In this context, pulsed laser ablation in liquid was performed according to the protocol as given above. Additionally, polyethylenimine was added to the liquid in the desired concentrations, in particular 10 μg/ml, 25 μg/ml, 50 μg/ml or 100 μ/ml, based on the liquid.
Preparation of Branched and Linear Polyethylenimine (PEI)
Branched PEI (Sigma Aldrich, 25 kDa) is a highly viscous solution. It was weighed, dissolved in PBS and adjusted to a 100 mg/ml stock solution. For use, stock solution was diluted to 1 mg/ml, filtered through a 0.22 μm membrane and stored at 4° C. The 10 kDa and 25 kDa linear PEIs (Polysciences Inc., Warrington, Pa., USA) were bought as powder and dissolved in water before using. To this end, the PEI was mixed with UltraPure distilled water at a concentration of 1 mg/ml and then heated to 80° C. until the solution was clear. The PEI solution was then cooled to room temperature and the pH value was adjusted to 7.0 using HCl. The PEI solution was then sterile filtered through a 0.22 μm membrane filter and stored at 4° C. The molecular weight of PEI has been determined by means of gel permeation chromatography or according to DIN 55672-3: 2016-03, respectively. Commercially available PEI variants jetPEI® and jetPEI®-hepatocyte (Polyplus Inc., Illkirch, FR) as well as Transporter5™ (Polysciences Europe GmbH, Hirschberg an der Bergstraße, DE) are delivered in a ready-to-use state.
Conjugation of Gold Nanoparticles with polyethylenimine by Admixing
In order to conjugate gold nanoparticles with polyethylenimine by admixing, the gold nanoparticles obtained by laser ablation have been incubated with the transfection reagent one day before transfection and dialyzed against ddH2O with a 50 kDa molecular weight cut off. The gold nanoparticles were diluted with ddH2O to a concentration of 160 μg/ml before using.
Conjugation of PEI-Conjugated Gold Nanoparticles with Nucleic Acid Molecules
Gold nanoparticles conjugated with transfection agents on the basis of polyethylenimine have been further conjugated with nucleic acid molecules by adding nucleic acid molecules in the desired amounts to the PEI-conjugated gold nanoparticles. In particular, further conjugation of the gold nanoparticles with nucleic acid molecules is performed immediately before transfection. In this context, reference can also be made to the further explanations regarding the transfection as such.
Vectors Designed for Further Expression Studies
The vectors, in particular the vectors as shown according to
Cell Cultures
For transfection analyses, the liver cancer cell lines HLF and HLE have been used. Both cell lines originate from human hepatocellular carcinoma. The HLF and HLE cells derived from the same patient have been obtained form the Riken Tissue bank in Japan. Furthermore, the cell line HT1080 has been used in order to analyze the transfection and expression in non-liver tissue, in particular fibroblasts. The cell line HT1080 is a human fibrosarcoma cell line (DMSZ, Braunschweig, Germany). In addition, transfection experiments have been transformed in rat hepatocytes. The cells were grown in Dulbecco's Eagle's Medium (DMEM) with 4,6 mM glucose and 2 mM GlutaMAX™ supplement with 10 wt.-% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. For antibiotic selection with the neomycin analogue geneticin (G418), the medium was supplemented with 1 mg/ml geneticin (commercially available from Gibco BRL, Thermo Fisher Scientific). All cells are adherent and form monolayers in culture; they have been split two to three times a week. For splitting, the cultures were washed with a solution on the basis of phosphate buffered saline (PBS, commercially available from Gibco BRL, Thermo Fisher Scientific) and incubated with Trypsin-EDTA until the monolayer dissociated. Cells were then transferred into new cell culture dishes based to their proliferation rate. Cells were grown at 37° C. in an atmosphere with 5 vol.-% CO2.
General Transfection Protocol
The transfection as such has been performed according to standard protocols. In particular, for transfection 200,000, 300,000 or 500,000 cells were seeded in 6-well tissue-culture plates. Cell counting of the different cell lines has been performed by using a Neubauer counting chamber. At the next day, cells were transfected with vector DNA using different transfection reagents. In this context, cells were cultured in 1 ml standard culture medium with the transfection reagent. 6 hours after transfection, standard medium was added to the cell culture wells. 24 hours after transfection, the medium was exchanged. After two or three days, GFP-expression was determined via Fluorescence-activated cell sorting (FACS) analysis.
Transfection with Polyethylenimine (Without Gold Nanoparticles)
For transfection with PEI as transfection reagent, DNA and PEI were separately diluted in 100 μl 150 mM NaCl. The PEI solution was then added to the DNA solution. The PEI/DNA solution was mixed, incubated for 15 minutes at room temperature and then added to the cells.
Transfection with Conjugated Gold Nanoparticles
HLF cells, HT1080 cells and rat hepatocytes were transfected with conjugated gold nanoparticles according to the present invention. The unconjugated gold nanoparticles had an average particle diameter of either 5 nm or 50 nm, determined by analytical disc centrifugation and transmission electron microscopy (TEM). Furthermore, different variants of PEI have been used, namely 25 kDa branched PEI (for example available from nanoComposix Europe, Prague, CZ), 25 kDa linear PEI (for example available from Nanopartz Inc, Loveland, Calif.), Transporter5™ (Polysciences Europe GmbH, Hirschberg an der Bergstraße, DE) and jetPEI®/jetPEI®-Hepatocyte (Polyplus Inc., Illkirch, FR). For the purpose of transfection with conjugated gold nanoparticles according to the present invention, the naked or unconjugated gold nanoparticles have been pre-incubated with the transfection reagent one day before transfection and dialyzed against ddH2O with a 50 kDa molecular weight cut off. The PEI-conjugated gold nanoparticles were diluted with ddH2O to a concentration of 160 μg/ml before using. Afterwards, the complexes of gold nanoparticles and polyethylenimine were further conjugated with the DNA by incubating them with the nucleic acid molecules for 2 to 5 minutes before adding to the cells for the purpose of transfection.
Fluorescence-Activated Cell Sorting (FACS)
FACS analyses were conducted to determine the number of GFP-expressing cells, as well as the mean fluorescent intensity (MFI) and the amount of non-apoptotic cells three days after transfection. In this context, cells were washed once with 2 ml phosphate buffered saline (PBS). Afterwards the cells were trypsinized with 0.5 ml Trypsin-EDTA (0.05 wt.-% Trypsin, 0.02 wt.-% EDTA) and the reaction was stopped by adding cell culture medium. The detached cells were transferred into a FACS tube and centrifuged for 5 min at 1,200 rpm. The supernatant was then removed and the cell pellet dissolved using PBS containing 2 wt.-% fetal calf serum (FCS) and 4′,6-diamidino-2-phenylindole (DAPI). For every FACS analysis a sample without DAPI-staining was furthermore analyzed. Data analysis was conducted using BD FACSDiva™ as software.
Factor Level Measurement
In order to determine the factor level, 24 hours after transfection, the cell culture medium was removed and the cells were cultured in 1 ml medium. After another 24 hours, the cell culture supernatant was collected and immediately frozen at −80° C. until factor level measurement was performed. During factor level measurement the amount of time, which is required for a plasma sample to clot, is recorded. Coagulation endpoints have been assessed by measuring changes in optical density with a turbidimetric method. All measurements were conducted using an ACL Top 500 (Werfen GmbH, Kirchheim near Munich, Del.).
With respect to the provision of conjugated gold nanoparticles for the use in an improved genetic approach for the treatment of monogenetic disorders, in particular haemophilia, studies with different malignant cell types and non-malignant rat hepatocytes have been performed. The results of the studies performed serve as a basis for the preparation of conjugated gold nanoparticles for the use in gene therapy and/or a nanoparticle-based delivery system for the use in gene therapy of monogenetic disorders.
Influence of the S/MAR Element on Transfection and Expression Efficiency
In order to establish an optimal S/MAR variant with respect to a long-term expression—i. e. episomal persistence—of the coding sequence in the target cells, in particular the liver or fibrous tissue, the long-term expression of GFP under different S/MAR variants in various cell types transfected with the afore described test vectors pEPI1-SM-L and pEPI1-SM-S (cf.
Transfection of Cell Lines
In order to test the influence of different S/MAR variants on the episomal persistence of nucleic acid molecules, liver cancer cells of the human hepatoma cell line HLF have been transfected with the afore-described vectors pEPI1-SM-S (
Test Procedure
The expression of GFP in the transfected cells was measured as an indicator for episomal persistence 24 hours after transfection. Afterwards, GFP expression in the cells was measured weekly. Since the malignant cell lines used for the test series are—in contrast to healthy liver cells, in particular hepatocytes, and healthy fibrous tissue cells—fast dividing cells, the test series were performed under short-term selection conditions on the basis of geneticin (G418) present for 2 weeks and long-term selection conditions on the basis of geneticin (G418) present over the whole observation period. In order to measure the expression of GFP, cells were harvested and analyzed by flow cytometry. In this context, the percentage of cells expressing GFP was determined. Furthermore, the MFI has been determined.
Results
The results of the transfection experiments regarding the influence of different variants of the S/MAR elements on episomal persistence are graphically depicted in
Influence of the Particle Size of the Gold Nanoparticles on Transfection Efficiency
Furthermore, the influence of the size, i.e. the average particle diameter, of the “naked” laser-ablated gold nanoparticles on the transfection efficiency of conjugated gold nanoparticles has been investigated. For this purpose, liver cancer cell lines HLF and HepG2 have been conjugated with conjugated gold nanoparticles on the basis of laser-ablated particles with a size of 5 nm or 50 nm, respectively. As transfection reagent, the particles comprised 25 kDa branched PEI and as nucleic acid molecules the vector pEPI-SM-S (cf.
Transfection of Cell Lines
With respect to transfection, 200,000 cells per well in a 6-well format have been transfected with conjugated gold nanoparticles on the basis of 30 μg gold nanoparticles with 18 μg of 25 kDa branched PEI and 20 μg nucleic acid molecules. As negative control, cells have been transfected without gold nanoparticles, wherein the same amount nucleic acid molecules and polyethylenimine has been used.
Test Procedure
Cells were analyzed for GFP expression three days after transfection by flow cytometry. In this context, the percentage of cells expressing GFP was determined.
Results
Influence of the Weight Related Ratio of DNA to polyethylenimine
According to the studies performed by applicant, the influence of the weight related ratio of the DNA to polyethylenimine in conjugated laser-ablated gold nanoparticles has been investigated. In this context, different variants of polyethylenimine (branched PEI and linear PEI, both with a molecular mass of 25 kDa) have been tested. Furthermore, different weight related ratios of transfection reagent to nucleic acid molecules of 1:1.1, 3:1, 6:1 and about 12:1 have been tested. The conjugated gold nanoparticles used for this test series comprised the vector pEPI-SM-S (cf.
Transfection of Cell Lines
200,000 cells (HLF or HT1080) per well of a 6-well plate have been transfected with conjugated gold nanoparticles on the basis of 30 μg laser-ablated gold nanoparticles with an average particle diameter of 5 nm, nucleic acid molecules in amounts of 0.7 μg, 1.5 μg, 3 μg or 10 μg, respectively, and polyethylenimine (either branched or linear PEI with a molecular mass of 25 kDa) in an amount of 9 μg.
Test Procedure
Cells were analyzed for GFP expression three days after transfection by flow cytometry. In this context, the percentage of cells expressing GFP was determined (
Results
Influence of the polyethylenimine Variant
According to the studies performed by applicant, the influence of the polyethylenimine variant in the conjugated gold nanoparticles according to the present invention on the transfection and expression efficiency has been investigated. In this context, different variants of polyethylenimine (25 kDa linear PEI, 10 kDa linear PEI, Transporter5™ and linear jetPEI®) have been tested as transfection reagent in conjugated gold nanoparticles. Furthermore, in this context the conjugated gold nanoparticles were tested with two different quantities of PEI and two different weight related ratios of transfection reagent to nucleic acid molecules.
Transfection Procedure
200,000 cells (HLF or HT1080) per well of a 6-well plate have been transfected with conjugated gold nanoparticles on the basis of 30 μg laser-ablated gold nanoparticles with a particle size of 5 nm, nucleic acid molecules in amounts of 1.5 μg, 3 μg or 6 μg and polyethylenimine (25 kDa linear PEI, 10 kDa linear PEI, Transporter5™ or linear jetPEI®) in an amount of 9 μg or 18 μg.
Test Procedure
Cells were analyzed for GFP expression three days after transfection by flow cytometry. In this context, the percentage of cells expressing GFP was determined (
Results
With respect to the results of studies in liver cancer cell line HLF, it can be seen from
With respect to the results of studies in HT1080 cells, it can be seen from
JetPEI® with a weight related ratio of polyethylenimine to nucleic acid molecules of 3:1. Except for linear polyethylenimine with a molecular mass of 10 kDa, higher amounts of polyethylenimine and nucleic acid molecules resulted in higher GFP expression levels for all tested variants of polyethylenimine. The results are further confirmed by the results of the determination of the mean fluorescence intensity (MFI) of eGFP in the GFP positive cells, which are depicted in
Comparison of Laser-Ablated and Chemically Synthesized Gold Nanoparticles
Furthermore, studies have been performed in order to compare the influence of the use of laser-ablated gold nanoparticles to the use of chemically synthesized nanoparticles on the transfection and expression efficiency. The respective studies have been performed in liver cancer cell line HLF and fibrosarcoma cell line HT1080. In this context, conjugated gold nanoparticles comprising either 10 kDa linear or 25 kDa branched PEI have been used. As nucleic acid molecules, the conjugated gold nanoparticles comprised the vector pEPI-SM-S (cf.
Transfection Procedure 200,000 cells (HLF or HT1080) per well of a 6-well plate have been transfected either with conjugated gold nanoparticles on the basis of laser-ablated gold nanoparticles with an average particle diameter of 5 nm, nucleic acid molecules in amounts of 1.5 μg, 3 μg or 6 μg and polyethylenimine (either 25 kDa linear PEI or 10 kDa linear PEI) in an amount of 9 μg or 18 μg or with chemically synthesized gold nanoparticles comprising 25 kDa linear PEI or 10 kDa linear PEI as transfection reagent and nucleic acid molecules in amounts of 350 ng, 1 μg, 3 μg, 6 μg, 9 μg or 20 μg.
Test Procedure
Cells were analyzed for GFP expression three days after transfection by flow cytometry. In this context, the percentage of cells expressing GFP was determined (
Results
With respect to the cell viability, it can be seen from
FISH-Analysis of Episomal Persistence
Furthermore, studies on the basis of fluorescence in situ hybridization have been performed in HLE cells in order to investigate the episomal persistence of the nucleic acid molecules, in particular the vector, transferred on the basis of conjugated gold nanoparticles according to the present invention.
Transfection Procedure
300,000 cells (HLE) per well of a 6-well plate have been transfected with conjugated gold nanoparticles on the basis of 30 μg laser-ablated gold nanoparticles, 18 μg of branched PEI with a molecular mass of 25 kDa and 6 μg of nucleic acid molecules (pEPI-SM-S, cf.
Test Procedure
Subsequent to transfection and cultivation, FISH analysis has been performed. After ten weeks of cultivation with an initial neomycin selection for two weeks, the cells were arrested in metaphases with colcemid and FISH analysis was performed with a biotin-labeled probe for detection of the GFP cDNA.
Results
Factor Level Measurements in HLF/HT1080 Cells
Furthermore, studies have been performed in order to investigate the factor activity of coagulation factor FIX after transfection of HLF and HT1080 cells with conjugated laser-ablated gold nanoparticles.
Transfection Procedure
300,000 cells/well (HT1080, HLF) in a 6-well format have been transfected with conjugated gold nanoparticles on the basis of 30 μg laser-ablated gold nanoparticles, 18 μg transfection reagent (either Transporter5™ or 10 kDa linear PEI) and 6 μg nucleic acid molecules (either pEPI1-SM-S according to
Test Procedure
With respect to the cultivation of the cells, the cell culture medium was exchanged 4 and 24 hours after transfection and cells were kept in culture for three additional days. Cell culture supernatants were collected to determine the FIX activity.
Results
The respective results are depicted in
Factor Level Measurements in Primary Rat Hepatocytes
Furthermore, rat hepatocytes have been transfected with conjugated laser-ablated gold nanoparticles in order to investigate the transfection efficiency, on the one hand, and factor activity level, on the other hand. In this context, stable amounts of laser-ablated gold nanoparticles have been used. Furthermore, two different amounts (9 μg or 18 μg) of the transfection reagent (Transporter5™, Polysciences Europe GmbH, Hirschberg an der Bergstraße, DE or 25 kDa linear PEI) and nucleic acid molecules in amounts of 3 μg or 6 μg have been used in the conjugated gold nanoparticles.
Transfection Procedure
500,000 cells/well in a 6-well format have been transfected with conjugated gold nanoparticles on the basis of 30 μg laser-ablated gold nanoparticles, 9 μg or 18 μg transfection reagent (25 kDa linear PEI or Transporter5™) and 9 μg or 18 μg nucleic acid molecules (pEGFPc1, coding for eGFP under the control of a CMV promoter; pCDNA3F9Pco, coding for FIX padua under the control of the CMV promoter, cf.
Test Procedure
With respect to cultivation, cell culture medium was exchanged 4 and 24 hours after transfection and cells were incubated for additional three days. Subsequently, supernatants were collected for FIX activity analysis and GFP-transfected cells were analyzed for GFP expression by flow cytometry. On the basis of flow cytometry, the percentage of GFP positive cells, the mean fluorescence intensity (MFI) value and the percentage of non-apoptotic cells have been determined with respect to cells transfected with a GFP expressing vector.
Results
On the basis of
Simultaneous Laser-Ablation and Conjugation
Furthermore, transfection studies have been performed in HLF cells and HT1080 cells in order to investigate the transfection efficiency of conjugated gold nanoparticles according to the present invention, wherein conjugation of the nanoparticles with polyethylenimine has been performed simultaneously with laser-ablation of the gold nanoparticles.
Preparation of PEI-conjugated gold nanoparticles
For this purpose, PEI-conjugated gold nanoparticles have been produced as described above according to the general experimental procedures. In this context, the buffer which has been used for pulsed-laser ablation in liquid contained different concentrations of branched polyethylenimine with a molecular mass of 25 kDa, namely concentrations of 10 μg/ml, 25 μg/ml, 50 μg/ml or 100 μg/ml. The gold nanoparticles as such had an average particle diameter of 5 nm, wherein conjugation during laser ablation increased the hydrodynamic diameter to a range of 14 to 22 nm, determined by dynamic light scattering.
Transfection Procedure
300,000 cells/well (HT1080, HLF) in a 6-well format have been transfected with conjugated gold nanoparticles on the basis of 30 μg laser-ablated and PEI-conjugated gold nanoparticles obtained as described above and 3 μg, 6 μg or 9 μg nucleic acid molecules (pEPI1-SM-S according to
Test Procedure
With respect to cultivation of the cells, 4 hours and 24 hours after transfection, the cell culture medium was exchanged and cells were kept in culture for additional three days. Thereafter, cells were collected and analyzed by flow cytometry in order to determine the percentage of GFP positive cells, the mean fluorescence intensities (MFI) and the percentage of non-apoptotic cells.
Results
The respective results with respect to the HLF cells are depicted in
The results with respect to the HT1080 cells are depicted in
Transfection Efficiency of Conjugated Gold Nanoparticles Comprising a Layer-By-Layer Assembly
Transfection studies have been performed in HLF and HT1080 cells in order to determine the transfection efficiency of conjugated laser-ablated gold nanoparticles comprising a layer-by-layer assembly with respect to the transfection reagents. In particular, it was analyzed whether a second layer of transfection reagent leads to higher transfection and/or gene transfer efficiencies.
Preparation of PEI-Conjugated Gold Nanoparticles
Laser-ablated gold nanoparticles with an average particle diameter of 5 nm have been prepared according to the general protocol for laser ablation. In a first step, the laser-ablated gold nanoparticles have been conjugated and/or coated with a first (inner) polyethylenimine layer comprising Transporter5™ (Polysciences Europe GmbH, Hirschberg an der Bergstraße, DE). After adding nucleic acid molecules on the basis of the vector pEPI-SM-S (cf.
Transfection Procedure
300,000 cells/well (HT1080, HLF) in a 6-well format have been transfected with the above described conjugated gold nanoparticles on the basis of 30 μg laser-ablated gold nanoparticles, 9 μg Transporter5™ as first (inner) transfection reagent, 3 μg nucleic acid molecules (pEPI-SM-S, cf.
Test Procedure
With respect to cultivation, the cell medium was exchanged 4 and 24 hours after transfection. Cells were kept in culture for two additional days and then analyzed by flow cytometry to determine the percentage of GFP expressing cells in order to determine the percentage of GFP positive cells, the mean fluorescence intensities (MFI) and the percentage of non-apoptotic cells.
Results
The results regarding HLF cells are depicted in
Promoter Activity of SERPINA1 and hAAT Promoters
Transfection studies in HLF and HT1080 cells have been performed in order to investigate and compare the activity of the promoters SERPINA1 and hAAT in different target cell types. For this purpose, the target cells have been transfected with either the vector peAATEG according to
Transfection Procedure
300,000 cells/well (HT1080, HLF) in a 6-well format were transfected by admixing 3 μg or 6 μg nucleic acids and 18 μg transfection agent (Transporter5™, Polysciences Europe GmbH, Hirschberg an der Bergstraße, DE) to the cells.
Test procedure
With respect to cultivation, the cell medium was exchanged 4 and 24 hours after transfection. Cells were kept in culture for two additional days and then analyzed by flow cytometry to determine the percentage of GFP expressing cells, the mean fluorescence intensity value (MFI) and the percentage of non-apoptotic cells.
Results
TEM-Analyses of Laser-Ablated Gold Nanoparticles
In order to compare 5 nm chemically synthesized and laser-ablated gold nanoparticles with respect to their surface properties and size distribution, analyses on the basis of transmission electron microscopy (TEM) have been performed before and after conjugation.
For the purpose of TEM-analysis, unconjugated laser-ablated particles (
Furthermore, TEM analyses have been performed after conjugating the particles with 25 kDa linear polyethylenimine as the ligand. For chemically synthesized gold nanoparticles, the transfection reagent was covalently bound to the surface of the nanoparticles by thiol groups. In contrast to this, the binding of the transfection reagent to the laser-ablated gold nanoparticles was based on electrostatic interactions. The TEM images (cf.
The current standard therapy for haemophilia comprises a life-long prophylactic administration of recombinant factors FVIII or FIX. However, frequent and expensive applications of the factors are necessary due to the short plasma half-life. On the basis of the present invention, a novel non-viral gene therapy approach for haemophilia B by transferring a normal copy of the mutated FVIII and/or FIX gene into the target cells, preferably hepatic cells, has been developed. This novel approach enables the target cells to produce the missing protein. Furthermore, this approach is applicable for any other monogenetic disorder associated with a lack of certain liver-specific or liver-expressed proteins due to a mutation coding for the gene of the respective protein.
According to the present invention, laser-ablated gold nanoparticles (AuNPs) as carriers for the vector DNA have been proven as superior over chemically produced gold nanoparticles with respect to the DNA transfer, compatibility and non-toxicity. Furthermore, the conjugated laser-ablated gold nanoparticles also are non-toxic, non-immunogenic and likely safer when compared to approaches with viral vectors.
With respect to the polyethylenimine, a particularly stable bond of the DNA to the gold nanoparticles as well as an efficient endosomal release of the DNA after cellular uptake has been achieved with linear PEI, preferably with a molecular weight of about 25 kDa.
Furthermore, a high-level production of clotting factors FVIII and/or FIX has been achieved by gene expression of the transgene under the control of different promoters, optimized for expression by in-/excluding introns, activating mutations and/or codon-optimization.
In order to further enhance specificity and efficiency of gene transfer, according to a particularly preferred embodiment of the present invention a layer-by-layer approach has been established, where two layers of PEI have been used. On this basis transfection efficiency is surprisingly increased. Furthermore, on the basis of such layer-by-layer approach or assembly the specificity of the conjugated gold nanoparticles with respect to the target cells can be improved. In particular, a layer-by-layer approach allows for cell specific targeting. In this context, an outer or second layer on the basis of a PEI variant that carries galactose residues (for example JetPEI®-Hepatocyte) to target the asialoglycoprotein receptor (ASGPR) has been proven suitable for an efficient targeting of the gold nanoparticles to hepatic cells or hepatocytes. Such second layer is not detrimental and can even increase gene transfer efficiency further.
Additionally, an improved method for the conjugation of laser-ablated gold nanoparticles has been found wherein conjugation is performed simultaneously with laser-ablation of the gold nanoparticles as such.
Finally, it was also found that vectors comprising the hAAT-promoter derived from human alpha-1 antitrypsin direct an efficient expression of coding sequences in different cell types, in particular liver cells and fibroblasts.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP2018/079997 | Nov 2018 | US |
| Child | 17155411 | US | |
| Parent | PCT/EP2018/070453 | Jul 2018 | US |
| Child | PCT/EP2018/079997 | US |
