EFFICIENT GENE DELIVERY TOOL WITH A WIDE THERAPEUTIC MARGIN

Abstract

The disclosure relates to novel saponins comprising acetyl residues on two of their sugar residues. These saponins are able to enhance the transfection efficiency to a high extent and show much less cytotoxic side effects than already known saponins.

Claims

1. A saponin according to formula (I): ##STR00008## wherein: R.sup.1 is independent from other R.sup.1 residues in the same molecule H or an acetyl residue, with the proviso that at least two R.sup.1 residues are acetyl residues; and R.sup.2 is independent from other R.sup.2 residues in the same molecule H or an acetyl residue, with the proviso that at least two R.sup.2 residues are acetyl residues.

2. The saponin according to claim 1, wherein it carries exactly four acetyl residues.

3. The saponin according to claim 2, wherein in each case one acetyl residue is bonded to the oxygen atoms in C3 and C4 position of the corresponding quinovose residue and to the oxygen atoms in C4 and C6 position of the corresponding glucose residue.

4. (canceled)

5. (canceled)

6. A medical method for delivering a nucleic acid, a lipid, a peptide and/or a protein to a human or animal by using a saponin according to claim 1 as transfection enhancing agent.

7. The medical method according to claim 6, wherein the saponin is applied in combination with at least one transfection reagent chosen from the group consisting of liposomal-based transfection reagents and polymer-based transfection reagents.

8. A transfection composition, comprising a saponin according to claim 1.

9. The transfection composition according to claim 8, further comprising at least one transfection reagent chosen from the group consisting of liposomal-based transfection reagents and polymer-based transfection reagents.

10. A method for an in-vitro transfection, comprising the step of incubating a cell with a nucleic acid in the presence of a saponin according to claim 1.

11. The method according to claim 10, wherein the cell is a eukaryotic cell.

12. The method according to claim 10 or 11, wherein the nucleic acid forms part of a nanoparticle.

13. The method according to claim 10, wherein the saponin is used in a concentration lying in a range of 1 μg/mL to 50 μg/mL.

14. The method according to claim 10, wherein the saponin is used in combination with at least one transfection reagent chosen from the group consisting of liposomal-based transfection reagents and polymer-based transfection reagents.

15. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Further details of aspects of the solution will be explained with respect to an exemplary embodiment and accompanying Figures.

[0058] FIG. 1A shows the results of cell viability tests with the saponin GE1741 at different saponin concentrations.

[0059] FIG. 1B shows the results of cell viability tests with the saponin SO1861 at different saponin concentrations.

[0060] FIG. 1C shows the results of cell viability tests with the saponin AG1856 at different saponin concentrations.

[0061] FIG. 1D shows the results of cell viability tests with different saponins at a saponin concentration of 24 μg/mL.

[0062] FIG. 2 shows the results of cell viability tests via impedance measurement with the saponin AG1856 at different saponin concentrations.

[0063] FIG. 3 shows the results of cell viability tests with different saponins at different saponin concentrations.

[0064] FIG. 4 shows the transfection efficiency of AG1856 for the transfection of nanoplexes comprising DNA bound to a peptide.

[0065] FIG. 5 shows the transfection efficiency of different saponins for the transfection of nanoplexes comprising DNA bound to a peptide.

[0066] FIG. 6A shows a first plot of the results of cell viability tests upon AG1856-mediated delivery of nanoplexes.

[0067] FIG. 6B shows a second plot of the results of cell viability tests upon AG1856-mediated delivery of nanoplexes.

[0068] FIG. 7 shows the results of an in vivo toxicity study.

[0069] FIG. 8 shows the results of an in vitro toxicity study.

DETAILED DESCRIPTION

Exemplary Embodiment: Isolation of the Saponin AG1856 from Agrostemma githago L

[0070] Seeds of Agrosternrna githago L. have been used for extraction (Agrostemmae semen, AGRO 26/80, from the Federal Center for Plant Breeding Research on Cultivated Plants (BAZ) in Gatersleben). Following grinding, 190 g of seeds were defatted overnight by Soxhlet extraction using petroleum ether. The defatted seed powder (177.1 g) was extracted three times using each 1 L 90% methanol. The methanol was evaporated by vacuum distillation and the water phase was freeze-dried (Christ alpha 2-4, Osterode, Germany). The yield of dry extract was 11.5 g (6%).

[0071] Size Exclusion Chromatography

[0072] The dry extract (200 mg) was dissolved in 1 mL DMSO and 1 mL of 50% methanol. This solution was subjected to size exclusion chromatography by medium pressure chromatography (MPLC, Azura-system, Knauer, Germany) equipped with a Sephadex™ LH-20 column (10×2000 mm). Elution was performed using methanol/water (1:1). The flow rate was 1 mL/min; detection was performed at 210 nm. Fractions were collected and dried by vacuum centrifugation and freeze-drying.

[0073] High Performance Liquid Chromatography (HPLC)

[0074] Selected fractions from the MPLC were subjected to HPLC (LC-8A Shimadzu) using a C18 column (Kinetex® 5 μm C18 100 A, LC Column 250×10.0 mm). The solvent system was (A) acetonitrile and (B) water (0.01% trifluoroacetic acid). A mobile phase gradient (45 min) was applied: 80 to 70% (B) over 10 min, then to 70 to 60% (B) between 10 and 15 min, 60 to 50% (B) between 15 and 20 min, 50 to 30% (B) between 20 and 25 min, 30 to 10% (B) between 25 and 30 min, 10 to 80% (B) between 30 to 40 min and maintained at 80% (B) for an additional 5 min. The flow rate was set at 2 mL/min, the analysis was recorded at 210 nm and 254 nm.

[0075] The structure of this saponin corresponds to formula (IV). While AG1856 comprises four acetyl residues (one in C3 position and one in C4 position of the quinovose residue as well as one in C4 position and one bound at the methyl residue in C6 position), preliminary data suggests that the amount and position of the acetyl residues can be varied within the indicated limits without significantly changing the properties of the respective saponin.

[0076] Testing the Properties of AG1856 in Comparison to Other Saponins

[0077] Murine neuroblastoma cells (Neuro2a cells, ATTC CCL-131™) were cultured in Dulbecco's Modified Eagle's Medium (DMEM), containing 1 g/L D-Glucose, 10% FBS and stable glutamine, at 37° C. and 5% CO.sub.2. These cells were then incubated with different saponins to test the cytotoxic effect of the saponins.

[0078] The tested saponins were GE1741, 501861 and AG1856. All 3 saponins share a common structural motive but bear slightly different sugar residues. This is elucidated in the following general formula (VII) with the subsequent Table 1.

##STR00007##

TABLE-US-00002 TABLE 1 Sugar residues of saponins GE1741, SO1861 and AG1856. Saponin R.sub.1 R.sub.2 R.sub.3 R.sub.4 GE1741 OH -QuiAcAc Xyl H SO1861 OH -QuiAc-(1.fwdarw.3)-Xyl Glc H AG1856 OH -QuiAcAc H GlcAcAc

[0079] The cell confluency as a measure of cell viability was determined optically via cell number calculating algorithms (Cytosmart). The results are depicted in FIGS. 1A to 1D.

[0080] FIG. 1A shows the results of an experiment in which different GE1741 concentrations were applied to Neuro2a cells 24 h after seeding. A distinct toxic effect was observed at a GE1741 concentration of 24 μg/mL. The initial peak was due to apoptotic cell expansion.

[0081] FIG. 1B shows the results of an experiment in which different SO1861 concentrations were applied to Neuro2a cells 24 h after seeding. A distinct toxic effect was observed at a 501861 concentration of 24 μg/mL. The initial peak was due to apoptotic cell expansion.

[0082] FIG. 1C shows the results of an experiment in which different AG1856 concentrations were applied to Neuro2a cells 24 h after seeding. No distinct toxic effect was observed at an AG1856 concentration of 24 μg/mL.

[0083] FIG. 1D shows a comparison of the different saponins GE1741, 501861 and AG1856 applied to Neuro2a cells at a concentration of 24 μg/mL. Only AG1856 showed no toxic effect at a concentration of 24 μg/mL.

[0084] Buffer was used in each case as negative control.

[0085] Cell Impedance Measurements

[0086] FIG. 2 shows the results of a cell viability test via impedance measurement with different AG1856 concentrations. The impedance (alternating current resistance) at the well bottom as real-time cell viability measure of adherent cells was determined by the iCELLigence® device. 8000 Neuro2a cells per well were seeded into two 8-well E-plates L8 and incubated for 24 h in a volume of 800 μL. For transfection toxicity studies 50 μL of reagent was added after the respective volume of culture medium was removed. Each 10 minutes the impedance/viability was measured. The results were analyzed and displayed with the RTCA Data Analysis Software.

[0087] The curves indicated in FIG. 2 represent the results of the following measurements:

TABLE-US-00003 Number of curve Tested compound 1 Negative control (buffer) 2 2 μg/mL AG1856 3 4 μg/mL AG1856 4 8 μg/mL AG1856 5 12 μg/mL AG1856 6 24 μg/mL AG1856 7 24 μg/mL GE1741 8 Positive control (4 μg/mL Puromycin)

[0088] The stepwise increase of AG1856 concentration led to no significant toxicity increase. In comparison, the severe toxicity of GE1741 (at a concentration of 24 μg/mL) prevented cell growth, similar to the toxic positive control puromycin (at a concentration of 4 μg/mL).

[0089] FIG. 3 shows the results of a cell viability test performed by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay with different AG1856 concentrations.

[0090] The determination of mitochondrial activity due to the formation of formazan out of MTT served as biochemical method to measure cell viability. A toxicity-mediated drop of viability could not be observed for AG1856 (curve 9), but was observed for GE1741 (curve 10) and 501861 (curve 11) in the same concentration.

[0091] FIG. 4 shows the ability of AG1856 in delivery of nanoparticles comprising DNA bound to a peptide (PD nanoplexes). The PD nanoplexes comprised DNA encoding for the green fluorescent protein (GFP). Therewith, the transfection efficiency could very easily be monitored by detecting the fluorescence of the cells that have been incubated with the corresponding DNA nanoparticles. The corresponding incubation period was chosen to be 48 hours in the present case.

[0092] Formulation of PD Nanoplexes

[0093] 20 mg of positively charged poly-lysine peptides without an integrin-receptor-targeting amino sequence were purchased from Genecust. The vector of p-EGFP-N3, coding for the green fluorescing protein (GFP), was obtained and propagated with DH5α— E.Coli cells (1.645 mg/mL). StemMACS™ eGFP mRNA (20 μg) and GFP encoding minicircle DNA (Gene Bank Accession: U55761) were used as further nucleic acids. In order to conduct the transfection, nanoplexes were formulated as follows: The poly-lysine peptides (P) and the p-EGFP-N3 vector (D), were diluted in water (each 50 μL) and mixed thoroughly by fast pipetting in a ratio of 4:1. The nanoplexes were allowed to form in a 30-minute incubation step. Thereafter, the nanoplex suspension was diluted with OptiMEM to a total volume of 1 mL. The commercial transfection reagent Lipofectamin®3000 was formulated as described by the manufacturer.

[0094] Sapofection (Transfection with Triterpene Saponins)

[0095] Neuro2a cells (15,000 cells/well) were seeded in a 24-well-plate with a well volume of 400 μL culture medium and incubated for 24 h. The transfection reagents were formulated as described above and admixed with saponin solution, if required. The culture medium was replaced with the transfection medium with a final amount of 500 ng DNA/RNA. After a 48 h incubation period, the transfection medium was removed, the cells were trypsinized and transferred in a polystyrene tube for flow cytometry (Cytoflex). For each measurement 10,000 cells were acquired. The transfection efficiency was determined by the analysis software Cyflogic (by comparison of the sample plots with the negative control in terms of fluorescence).

[0096] AG1856 showed already at a concentration of 2 μg/mL a very good transfection efficiency that was comparable to that of the gold standard Lipofectamin. At concentrations of 4 μg/mL or higher, the transfection efficiency of AG1856 was even higher than that of Lipofectamin. Data in FIG. 4 is expressed as mean±standard deviation (SD). Lipofectamin3000 was the positive control (N=4; p value<0.01 (**), p value<0.0001 (****), ns: non-significant compared to PD with one-way ANOVA).

[0097] FIG. 5 shows the transfection efficiency of high concentrated saponin-mediated transfections. Neuro2a cells were transfected with GFP-plasmid bearing nanoplexes as explained above with and without saponin co-application. The saponins GE1741, 501861 and AG1856 were used in their working concentrations or in a concentration of 24 μg/mL. The working concentration of GE1741 and 501861 presents the highest non-toxic concentration. For AG1856, it represents the lowest effective concentration, wherein an increase shows no significant transfection efficiency increase (cf. FIG. 4). While an increased concentration of 24 μg/mL led to a severe efficiency drop of GE1741- and SO1861-mediated transfections due to their toxicity, an even increased efficiency could be observed in case of AG1856-mediated transfections. Lipofectamin3000 served as positive control. (N=3; p value<0.01 (**), p value<0.0001 (****), ns: non-significant compared to PD with one-way ANOVA).

[0098] FIGS. 6A and 6B show cell viability tests upon AG1856-mediated delivery of GFP-minicircle-DNA-or RIP-minicircle-DNA-bearing nanoplexes. The purpose of the conducted experiment was to reveal the capability of performing non-toxic and highly efficient gene delivery as well as to exhibit the feasibility not just to deliver reporter genes, but therapeutic genes for tumor therapies.

[0099] FIG. 6A shows the viability upon transfection of GFP minicircle DNA, saporin minicircle DNA and dianthin minicircle DNA. Saporin and dianthin are cytotoxic proteins. If the transfection is successful, the transfected cells will die.

[0100] The curves depicted in FIG. 6A represent the results of the following measurements:

TABLE-US-00004 Number of curve Tested compound 12 Negative control (buffer) 13 4 μg/mL AG1856 14 PD(dianthin minicircle, 1 μg) 15 PD(saporin minicircle, 1 μg) 16 PD(GFP minicircle, 1 μg) 17 PD(GFP minicircle, 1 μg) + 4 μg/mL AG1856 18 PD(saporin minicircle, 1 μg) + 4 μg/mL AG1856 19 PD(dianthin minicircle, 1 μg) + 4 μg/mL AG1856

[0101] No distinct toxicity of GFP minicircle DNA without and with AG1856 co-application could be observed. The transfection of nanoplexes with the ribosome inactivating proteins dianthin and saporin (dianthin minicircle DNA or saporin minicircle DNA) showed only severe toxicity when AG1856 was co-administered. AG1856 alone showed no toxicity.

[0102] FIG. 6B indicates that the transfection with minicircle DNA (being smaller, less degradable and more efficient than a DNA plasmid) coding for GFP-DNA results in a highly efficient gene delivery, when performed with AG1856. Four cells out of five were fluorescing as determined by flow cytometric analysis (n>3).

[0103] Further experiments confirmed the low toxicity of AG1856 in vivo and in vitro. These experiments will be explained in the following.

[0104] Chronic Toxicity Study of AG1856 In Vivo in Combination with Targeted Anti-Tumor Nanoplasmids

[0105] Objectives

[0106] This study was performed to evaluate the tolerability of combined treatment of nanoplasmids (NP) and AG1856 in Naval Medical Research Institute (NMRI) nu/nu mice.

DESCRIPTION

[0107] For the study, female NMRI nu/nu mice were used. Animals were treated with NP+AG1856 at doses of 50 and 90 μg/mouse respectively and after animal recovery in a second round at same doses.

Summary of Results and Discussion

[0108] The study revealed no side effects for the mice treated with the combination of NP+AG1856. Therefore, all repeated treatments were well tolerated. No changes in body weight were seen, cf. the results depicted in FIG. 7.

CONCLUSIONS

[0109] In conclusion, this treatment is well applicable for future therapeutic experiments at a given dose and application schedule. The application of 90 μg per mouse shows the superior tolerance of AG1856 even in high doses compared to other saponins.

[0110] Toxicity Studies of AG1856 In Vitro in Combination with Different Targeted Anti-Tumor Nanoplasmids

[0111] Material and Methods

[0112] To evaluate an enhancement of the transfection efficiency through AG1856 an optical based cytotoxicity assay after transfection with ribosome inactivating proteins (RIPs) was chosen.

[0113] Plasmids

[0114] As transgenes, 6-His-Agrostin RNA3 (Weise et al. 2020) and 6-His-Saporin 3 (Fabbrini et al. 1997) were cloned via BamHI and XbaI (Agrostin) and via SalI and NheI cleavage into pMC.CMV-MCS-SV40polyA (BioCat GmbH, Heidelberg, Germany) to produce minicircle with ZYCY10P3S2T (Kay et al. 2010). In addition to this, Saporin 3 and Gypsophillin S (Kokorin et al. 2019) were cloned into NTC9385R-BGHpA-Nanoplasmids and synthesized by Nature Technology Corporation (Lincoln, NB, USA) as previously described (Luke et al. 2011).

[0115] Nanoplex Formulation

[0116] For 3 wells (96-Well-Plate), 300 ng plasmid DNA were diluted in 25 μL water and mixed with 25 μL of a solution with 2,100 ng Peptide Y (K.sub.16GACYGLPHKFCG) (GeneCust, Dudelage, Luxembourg). After an incubation period of 30 min, the nanoplexes were formed automatically.

[0117] Cell Culture

[0118] Murine neuroblastoma cells Neuro2A (ATCC® CCL-131) were cultivated in BioWhittaker® Dulbecco's Modified Eagle's Medium (DMEM) (Lonza Group, Basel, Switzerland) supplemented with 10% FBS and 1% non-essential amino acids (Lonza Group, Basel, Switzerland) in a 5% CO.sub.2 atmosphere and 37° C.

[0119] Transfection

[0120] 4,000 cells per well were seeded in clear 96-well-plates and cultivated for 24 h. After this, the medium was exchanged against a mixture of freshly formulated nanoplexes (described above), culture medium (described above) and AG1856 (5 μg/mL) or water for the control group. This leads to 200 μL medium per well with 500 ng/mL DNA. As negative control, water was used and as positive control 5 μg/mL puromycin (Carl Roth, Karlsruhe, Germany). After the transfection, the cells were observed for 48 h.

[0121] Live Cell Imaging

[0122] The cytotoxic effect of the different constructs on the cells was measured by a CytoSMART® Omni, a camera-based system which took a photo of the bottom of each well every hour. The CytoSMART® Omni software calculated the confluence which is formed by living cells which are attached to the bottom. So, the cell growth could be monitored for 72 h.

[0123] Results and Discussion

[0124] The confluence values are normalized to the certain point of intervention to have a better comparability between the groups (Normalized Cell Index, NCI). After the intervention, all wells treated with nanoplexes but not with AG1856 showed no sign for inhibition of proliferation. They behaved like the negative control group and partially showed even a better growth. Although the cells which were treated with just AG1856 proliferated 12% worse than the negative group, the cytotoxic effect of the groups which were intervened with nanoplexes and

[0125] AG1856 was much bigger. The cells showed no more proliferation after 36 h. This indicates that the transfection was successful because after this period the RIPs were formed and inhibited the protein biosynthesis, which led to a cytostatic effect. The positive control behaved nearly equally but had no lag period before a cytostatic effect could be observed. The reason for that is that puromycin did not have to be formed and could inhibit the growth of the cells instantly.

[0126] FIG. 8 shows the detailed results, namely the confluence normalized on the point of intervention (t=24 h). The curves represent the confluency after treatment with the following constructs:

TABLE-US-00005 Number of curve Construct used for treatment 20 Minicircle Saporin 3 21 Nanoplex Saporin 3 22 Minicircle Agrostin RNA3 23 Nanoplex Gypsophilin S 24 negative control 25 AG1856 only 26 Minicircle Saporin 3 + AG1856 27 Minicircle Agrostin RNA3 + AG1856 28 Nanoplex Saporin 3 + AG1856 29 Nanoplex Gypsophilin S + AG1856 30 positive control 31 positive control + AG1856

[0127] All groups treated with RIP-nanoplexes and AG1856 showed strong retention in cell growth while the intervention on the groups which were treated just with nanoplexes and no AG1856 had no impact on the cell growth.

CONCLUSION

[0128] AG1856 was crucial for the cytotoxic effect of the RIP-coding nanoplexes. Without this transfection enhancer no cytotoxic effect could be observed.

LITERATURE CITED WITH RESPECT TO THE TOXICITY STUDIES

[0129] M. S. Fabbrini, E. Rappocciolo, D. Carpani, et al., Characterization of a saporin isoform with lower ribosome-inhibiting activity, The Biochemical journal, 322 (Pt 3) (1997) 719-727. [0130] M. A. Kay, C. Y. He and Z. Y. Chen, A robust system for production of minicircle DNA vectors, Nature Biotechnology, 28 (2010) 1287-1289. [0131] A. Kokorin, C. Weise, S. Sama, et al., A new type 1 ribosome-inactivating protein from the seeds of Gypsophila elegans M.Bieb, Phytochemistry, 157 (2019) 121-127. [0132] J. M. Luke, J. M. Vincent, S. X. Du, et al., Improved antibiotic-free plasmid vector design by incorporation of transient expression enhancers, Gene Therapy, 18 (2011) 334-343. [0133] C. Weise, A. Schrot, L. T. D. Wuerger, et al., An unusual type I ribosome-inactivating protein from Agrostemma githago L, Scientific Reports, 10 (2020) 15377.