Nanostructured lipid carriers, stable emulsions, and their uses

Abstract

Provided herein are nanostructured lipid carrier compositions, methods for making the same, and methods for using the same. The compositions comprise nanostructured lipid carriers (NLCs), which contain an oily core composed of a mixture of liquid-phase lipids and solid-phase lipids, a cationic lipid, a sorbitan ester, and a hydrophilic surfactant, and optionally a bioactive agent. The bioactive agent can be conjugated to the NLC. The compositions enable the delivery of biomolecules to cells for the induction of an immune response, e.g., for vaccine, therapeutic, or diagnostic applications. Also provided are compositions for stimulating an immune response, methods for making the same, and methods for using the same. [Selected Figure] Figure 1

Description

[Technical Field]

[0001] Cross-reference of related applications This application is based on U.S. Provisional Patent Applications No. 62 / 520,204 filed on June 15, 2017, No. 62 / 540,973 filed on August 3, 2017, No. 62 / 556,291 filed on September 8, 2017, No. 62 / 563,544 filed on September 26, 2017, No. 62 / 582,859 filed on November 7, 2017, and No. 62 / 62 / 62,204 filed on January 26, 2018. Claiming the benefit of priority under U.S. Provisional Patent No. 22,748, No. 62 / 622,755 filed on 26 January 2018, No. 62 / 669,262 filed on 9 May 2018, No. 62 / 677,336 filed on 29 May 2018, and No. 62 / 680,454 filed on 4 June 2018, the entire contents of each U.S. Provisional Patent Application are invoked by reference for all purposes.

[0002] Technical field Overall, this disclosure relates to the pharmaceutical and vaccine formulation fields. [Background technology]

[0003] Nucleic acid immunization is an attractive strategy for rapidly developing vaccines against existing or emerging infectious disease threats. Nucleic acid vaccine candidates can be readily synthesized using conventional methods and constructed within weeks of the emergence of a new infectious disease. In addition, because the biophysical characteristics of nucleic acid vaccines are independent of the expressed antigen, the development of antigen-specific processes required for the manufacture of these new vaccines is minimal. Currently, plasmid DNA vaccines are being developed for a select few infectious diseases, but so far, no DNA-based vaccines have been approved for human use due to associated complications (McKay, Cope et al. 2014, Tregoning and Kinnear 2014).

[0004] RNA-based platforms for antigen delivery have been proposed as a promising alternative to DNA-based platforms. The transient nature of RNA is advantageous in antigen delivery, meaning that the risk of long-term vaccine persistence is reduced compared to DNA, and furthermore, the delivered vaccine does not need to translocate to the nuclear nucleus for protein production. However, the relative instability of RNA and the finiteness of expression from a single mRNA transcript make large-scale distribution and use of these vaccines difficult in this field, and commercial development is also challenging. Much research has been done on methods to improve RNA stability through structural modification of RNA, and several solutions have been provided to this problem (Tavernier, Andries et al. 2011, Youn and Chung 2015). In particular, self-amplifying RNA-based vaccines have shown a promising mechanism for improving the scale and duration of antigen expression (outlined in Vander Veen, Harris et al. 2012, Ljungberg and Liljestrom 2015).

[0005] To enable a robust immune response, liposome formulations and oil-in-water emulsion formulations are typically used to enhance RNA delivery into cells (Geall, Verma et al. 2012, Ulmer, Mason et al. 2012, Brito, Chan et al. 2014, Bogers, Oostermeijer et al. 2015, Brito, Kommareddy et al. 2015, Geall and Ulmer 2015). In addition, these formulations may be used to enhance the intracellular delivery of drugs or other therapeutic substances. However, liposomes such as cationic lipid emulsions (CNEs) and oil-in-water emulsions are structurally unstable in physiological environments, which can increase the potential for toxicity from acute exposure to individual components. Furthermore, the potential toxicity of such carriers exacerbates or conflates the toxicity concerns generally associated with cationic phospholipids required for RNA adsorption (Bertholet et al. 2010). In addition, there is limited understanding of how the physicochemical properties of oil-in-water emulsions (e.g., size, surface charge, chemical properties of excipients and their relative ratios) affect RNA binding, delivery, and final antigen expression.

[0006] In other words, there is a need for a versatile, stable, and safe formulation platform that functions well both physically and chemically as a system for delivering bioactive agents, including nucleic acids, to cells.

[0007] All references cited herein, including patent applications and patent publications, are incorporated herein by reference in whole, as if each individual reference were explicitly and individually indicated to be invoked by reference. [Overview of the project]

[0008] The inventors have developed a formulation that is remarkably effective in the cellular delivery of bioactive agents. Accordingly, in particular, such a formulation (also referred to as a composition) and its use are provided herein. This formulation is a nanostructured lipid carrier (NLC) based formulation. It will be understood by those skilled in the art that NLC is composed of NLC particles. NLC is described in Beloqui et al., Nanomedicine. NBM 2016; 12:143-161. An exemplary NLC particle of the present invention comprises (a) an oily core containing liquid-phase lipids and solid-phase lipids, (b) a cationic lipid, (c) a hydrophobic surfactant (preferably a sorbitan ester (e.g., sorbitan monoester, sorbitan diester, or sorbitan triester)), and (d) a hydrophilic surfactant. The exemplary composition is stable and enables the cellular delivery of bioactive agents. The delivery of bioactive agents may be aimed, for example, at inducing an immune response and / or treating a disease or health condition in a subject.

[0009] These and other aspects of the present invention will become apparent by reference to the following detailed description and accompanying drawings. Furthermore, various references are listed herein, which describe specific aspects of the present invention in more detail and are therefore incorporated herein by reference in their entirety. [Brief explanation of the drawing]

[0010] [Figure 1-1] Figures 1A to 1E show a comparison of stability between CNE and NLC formulations. The figures compare the dynamic light scattering (DLS) particle size (Malvern Zetasizer Z / ZS) of the CNE formulation (QG386) and the NLC formulation (QG752) stored at 5°C (Figure 1A), 25°C (Figure 1B), 37°C (Figure 1C), 60°C (Figure 1D), and 80°C. Arrows are shown simply to help distinguish the lines. [Figure 1-2] Same as above.

[0011] [Figure 2]Figures 2A and 2B show the z-average particle size of NLC as a function of the oil / surfactant ratio or surfactant / oil ratio measured using dynamic light scattering (DLS).

[0012] [Figure 3-1] Figures 3A to 3E illustrate an optical concentration measurement method called gel delay assay (GRA). Figure 3A shows the amount of RNA bound to NLC as a function of the nitrogen / phosphate (N / P) ratio. Figures 3B to 3E show superimposed in vitro SEAP expression (relative luminescence, RLU) and RNA-NLC binding curves as a function of N:P values ​​for QG942, QG963, QG807, and QG843 (also known as CNE). [Figure 3-2] Same as above. [Figure 3-3] Same as above.

[0013] [Figure 4] Figures 4A to 4C show a comparison of DLS particle size (Z-mean, nm) of the formulation-RNA complex when the nitrogen / phosphate (calculated) ratio (Figure 4A), RNA / particle (theoretical) ratio (Figure 4B), or DOTAP / RNA mass ratio (calculated) (Figure 4C) is varied.

[0014] [Figure 5] Figure 5 shows the neutralizing antibody titers obtained from mice (n=5 / group) 14 days after single intramuscular administration of Zika antigen-expressing rvRNA complexed with different amounts of QG768 formulation. Neutralizing antibody titers were determined by the 80% plaque reduction neutralization test (PRNT80). Significance was confirmed by one-way ANOVA.

[0015] [Figure 6]Figure 6 shows the neutralizing antibody titers obtained from mice (N=5 / group) after intramuscular (IM) administration of RNA either conjugated with the formulation (0.1 μg of RNA mixed with the formulation in a 1:1 (v / v) ratio) or alone (naked) (0.1 μg or 10 μg of RNA mixed with physiological saline in a 1:1 ratio). Neutralizing antibody titers were determined by the 80% plaque reduction neutralization test (PRNT80). Significance was confirmed by one-way ANOVA.

[0016] [Figure 7] Figures 7A to 7E show the levels of chemokine release from human blood cells in the presence of the indicated formulations. Chemokine expression levels were determined by a Luminex assay using a multiplex cytokine bead array.

[0017] [Figure 8] Figures 8A and 8B show the in vitro rvRNA delivery ability of the selected formulations. 293T cells or BHK cells were transfected with 10 ng (Figure 8A) or 100 ng (Figure 8B) of SEAP-coding rvRNA using the indicated formulations. Lipofectamine was used as a positive control, and 10% sucrose solution as a negative control. After transfection, the supernatant was collected, and SEAP activity was measured using luminescence assay.

[0018] [Figure 9] Figure 9 shows the in vivo protein expression dynamics of the selected formulations. C57BL / 6 mice were injected 1 μg intramuscularly with rvRNA encoding SEAP, formulated in 10% sucrose, CNE, QG807, or QG808. Mice injected with physiological saline were used as mock controls. Serum was collected 3, 7, 14, 21, and 28 days after injection, and SEAP activity was measured using luminescence assay.

[0019] [Figure 10]Figure 10 shows the optimization of the RNA load of formulation QG807. The rvRNA encoding SEAP was diluted with 10% sucrose to various concentrations shown on the X axis and complexed with QG807 in a 1:1 ratio. Next, the formulated RNA was diluted to doses of 1 μg or 0.1 μg per 50 μL volume. Each dose was then injected intramuscularly into C57BL / 6 mice, and serum was collected 3 and 8 days after injection. Next, SEAP activity was measured by luminescence assay.

[0020] [Figure 11-1]Figures 11A to 11C. In Figures 11A and 11B, "mock" refers to 10% sucrose, "naked" refers to 100 ng of unformulated SEAP rvRNA, "CNE" refers to SEAP rvRNA complexed with CNE (containing Span 85), "QG768" refers to NLC containing Span 60, "QG906" refers to NLC containing Span 80, "QG808" refers to NLC containing Span 85, and "SQ807" refers to NLC containing Span 60. Figure 11A shows a comparison of four NLC formulations with different emulsifier compositions and CNE or diluents in terms of their ability to enhance protein expression and subsequent complex formation with RNA encoding secreted alkaline phosphatase (SEAP). Serum was collected from mice three days after a single 100 ng intramuscular (IM) injection (n=3 / group), and SEAP activity was measured. Each data point is plotted, and their mean ± standard deviation (SD) is shown. Figure 11B shows the same formulation used in Figure 11A, which formed a complex with RNA encoding ZIKV prM and E. Neutralizing antibodies were measured 14 days after a single 100 ng intramuscular injection (n=5 / group). Data analysis was performed using Tukey's multiple comparison test after one-way ANOVA (*p<0.05; NLC Span60 compared to CNE or NLC Span85, **p<0.008; NLC Span60 compared to NLC Span80, ***p=0.0007; CNE compared to NLC Span80 or Span85, no significant difference). Figure 11C shows CNE or NLC, composed of two different hydrophobic surfactants (Span 85 and Span 60), and neutralizing antibodies, which formed complexes with RNA encoding ZIKV prM and E, measured 14 days after a single 100 ng intramuscular injection. Data were analyzed by Tukey's multiple comparison test after multivariate two-way ANOVA. **p<0.0001. [Figure 11-2] Same as above. [Figure 12-1]Figures 12A-12H: C57Bl / 6 mice (n=9 / group) were inoculated with 1 μg of CNE-formulated RNA or NLC-formulated RNA by single intramuscular injection and compared with 10 or 1 μg of unformulated RNA (Figure 12A). Neutralizing antibodies were evaluated 14 days after inoculation. Subsequently, 3 mice per group were boosted with a second dose of each vaccine 28 days later (Figures 12B-12F). 46 days after boosting, the spleen was removed and stimulated with CD8 peptides corresponding to the epitopes in prM and E of ZIKV. Figures 14G-14H: Mice immunized with a single intramuscular dose (n=6 / group) were challenged with a lethal dose of ZIKV on day 30 after blocking the interferon-α receptor with monoclonal antibody administration. Serum for the virus was evaluated by plaque assay 4 days after the challenge (Figure 12G). Survival rates were monitored (Figure 12H). NLC4 was QG807. [Figure 12-2] Same as above.

[0021] [Figure 13] Figures 13A-13B. C57BL / 6 mice (n=3 / group) were intramuscularly injected with 1 μg of either unformulated SEAP RNA or SEAP RNA formulated with QG807, QG924, QG925, QG941, or QG942 by forming a complex with 400 μg / mL of RNA. SEAP expression was compared to that of QG807, which formed a complex at the optimal RNA concentration of 40 μg / mL (Figure 13A). BHK cells were incubated with each high RNA-load formulation diluted 1:20 for 4 hours, and then stained with propidium iodide and annexin to detect dead cells or cells undergoing apoptosis. Subsequently, the proportion of cells that were neither dead nor apoptotic was quantified using flow cytometry (Figure 13B).

[0022] [Figure 14] Figure 14 shows in vivo SEAP expression data, comparing QG807 complexed with 400 μg / mL or 40 μg / mL RNA.

[0023] [Figure 15-1] Figures 15A to 15D show the levels of chemokine release from human blood cells as a whole in the presence of the indicated formulations. Chemokine expression levels were determined by a Luminex assay using a multiplex cytokine bead array. [Figure 15-2] Same as above.

[0024] [Figure 16-1] Figures 16A-16D. Figure 16A shows exemplary NLC particles. Figure 16B shows the intensity-weighted size distribution of NLCv1 and CNE (QG386). Figure 16C shows the evolution of particle size (z-mean particle size) over 9 months to evaluate the colloidal stability of formulations stored at 25°C. Comparison of dynamic light scattering (DLS) particle size (z-mean particle size, Malvern Zetasizer Z / ZS) between CNE (QG386) and NLCv1 formulation (QG752). Figure 16D shows RNase protection by NLC containing a relatively small amount of Tween80 fraction in the surfactant phase (35% of the total mass of surfactant and cationic lipids), protecting rvRNA from degradation. [Figure 16-2] Same as above.

[0025] [Figure 17] Figure 17 shows an exemplary NLC manufacturing process.

[0026] [Figure 18]Figures 18A and 18B show the persistence of the neutralizing antibody response after two independent experiments with one or two doses of NLC-formulated ZIKV rvRNA. C57BL / 6 mice (n=5 / group) were inoculated with 1 μg or 0.1 μg of NLCv1-formulated ZIKV rvRNA via the intramuscular route once on day 0 (Figure 18A) or on days 0 and 28 (Figure 18B). Neutralizing antibody titers at various time points were compared to mice inoculated with 10 μg or 1 μg of unformulated (single) ZIKV rvRNA. Data are plotted as the mean ± standard deviation for each biological replica. The data from Figures 18A and 18B were log-10 transformed and analyzed by Tukey's multiple comparison test after one-way ANOVA, comparing the mean PRNT80 at each time point for each group with the peak antibody titers at day 14. Figure 18A compares NLCv1 (1 μg) or NLCv1 alone (10 μg) at day 88 and day 156 with antibody titers at day 14 (**p<0.0001, **p<0.001). Figure 18B compares NLCv1 (1 μg) at day 126 and day 209 with antibody titers at day 14 (*p=0.05, ***p=0.0001, respectively).

[0027] [Figure 19] Figures 19A-19B. Mice were inoculated with NLC-formulated RNA by intramuscular injection, and CD+ T cell levels were compared with those of 10 μg or 1 μg of unformulated RNA and CNE at day 14 (Figure 19A) and day 49 (Figure 19B).

[0028] [Figure 20-1]Figures 20A to 20E. Mice were inoculated with the indicated dose of NLC-formulated RNA by intramuscular injection. NLCv2 (QG942) was complexed with rvRNA encoding ZIKV prM / E with an N:P value of 15. C57BL / 6 mice were administered 100ng, 30ng, 10ng, and 3ng doses by single intramuscular injection (n=14 / group). Blood samples were collected 14 days later, and neutralizing antibody titers were evaluated using PRNT80 (Figure 20A), or the mice were euthanized (n=4 / group) and the percentage of antigen-specific B220loCD8+IFNγ+ T cells in total splenocytes was quantified (Figure 20B). These results were compared to mock-vaccinated mice (base), or 100ng or 10ng of rvRNA alone (sucrose) (n=14 / group), or 100ng of rvRNA formulated with NLCv1 (n=14) or CNE (n=4) at an N:P value of 15 (Figures 20A-20B). Data are shown as individual values ​​and as mean ± standard deviation.

[0029] Thirty days after vaccination, the remaining 10 mice / group were subjected to type I interferon antibody blockade as described in Smith et al., PLoS Negl Trop Dis. 11(1):e0005296 (2017), followed by 5 log 10 Mice were challenged with PFU's ZIKV Dakar 41525 strain, and blood samples were collected 4 days later to quantify the virus (viremia) in the blood using a plaque assay (Figure 20C). Data are shown as individual values ​​and as mean ± standard deviation. Survival rate (Figure 20D) and weight loss (Figure 20E) were monitored daily in mice. Data in Figure 25E are shown as mean ± standard deviation. Data from Figures 20A and 20C are log 10 The transformed data was analyzed using Tukey's multiple comparison test after one-way ANOVA (PRNT, Figure 25A). 80 Antibody titers were measured using the control group (mock), unformulated antibody titers (100 ng and 10 ng), and NLC. v2 Between the 100ng, 30ng, and 10ng dosage formulations: **** p<0.0001, or between p<0.0001 and CNE formulation 100ng * p=0.04;NLCv2 Between the 100 ng and 30 ng doses of the formulation and the 100 ng CNE formulation ** p = 0.006; The viral load in the blood in Figure 20C was compared among the mock control group, the non-formulated 100 ng and 10 ng doses, and the NLC v2 formulation or NLC v1 and all doses of the formulation, **** p < 0.0001). The data in Figure 20E were analyzed by Tukey's multiple comparison test after two-way ANOVA (change rate of body weight from day 5 to day 10, between the mock control group, or the 100 ng and 10 ng rvRNA alone, and all formulation groups, * p < 0.05). The data in Figures 20A - 20C are representative of two independent experiments. [Figure 20-2] The same as above. [Figure 20-3] The same as above.

[0030] [Figure 21-1] Figures 21A - 21D show the design and quality assurance of ZIKV rvRNA. Figure 21A shows a plasmid DNA encoding a Venezuelan equine encephalitis replicon derived from the vaccine strain TC-83 under the control of the T7 RNA polymerase promoter. Untranslated region (UTR), subgenomic region (SG), gene of interest (GOI). Figure 21B shows the design of a replicating viral RNA encoding the pre-membrane (prM) gene and envelope (E) gene of the ZIKV H / PF / 2013 strain, or secreted human embryonic alkaline phosphatase (SEAP). The supernatants of 293T cells transfected with ZIKV rvRNA or SEAP rvRNA were sedimented in 30% sucrose and then analyzed by Western blot (Figure 21C) using an anti-ZIKV polyclonal antibody and / or transmission electron microscopy (Figure 21D). [Figure 21-2] The same as above.

[0031] [Figure 22-1]Figures 22A–22H illustrate the physical relationship between NLCv2 and rvRNA, along with biological reactions including gene expression, immunogenicity, and reactogenicity. For in vitro experiments (Figures 22A–22D), NLCv2 was complexed with SEAP rvRNA at various N:P ratios. Gene expression (Figure 11A) was measured using the SEAP assay described above, particle size (Figure 11B) and zeta potential (Figure 22C) were measured by dynamic light scattering, and RNA binding rate (Figure 22D) was measured by densitometry after gel electrophoresis. The data in Figures 22A–22D are shown as mean ± standard deviation and are representative of at least three independent experiments. In in vivo experiments using C57BL / 6 mice (Figures 22E-22G), NLCv2 was complexed with ZIKV rvRNA or SEAP rvRNA in N:P ratios of 3, 5.6, 15, and 37. Mice (n=3 / group for SEAP, or n=5 / group for ZIKV) were injected via the intramuscular route with 1000 ng, 100 ng, or 10 ng of SEAP rvRNA or 1000 ng or 100 ng of ZIKV rvRNA. SEAP expression was measured by quantitative SEAP analysis 3 days after injection (Figures 22E-22G), and ZIKV neutralizing antibody titers were measured by PRNT80 14 days after injection (Figures 22F-22G). To measure reactogenicity (Figure 22H), guinea pigs (n=4 / group) were injected intradermally with 50 μg of ZIKV rvRNA complexed with NLCv2 at N:P values ​​of 3, 5.6, 15, and 37, and the redness diameter was measured 24 hours later. The SEAP data in Figures 22E to 22G are presented as mean ± standard deviation and represent data from a single experiment. On the other hand, the PRNT80 data in Figures 22F and 22G are presented as mean ± standard deviation and minimum / maximum box plots and represent data from three independent experiments. The data in Figure 22H are from a single experiment and represent individual biological replicas with mean ± standard deviation and minimum / maximum box plots. The Log10 transformation of the PRNT80 data in Figure 22G was analyzed using one-way ANOVA and Tukey's multiple comparison test (no significant difference between titers with N:P value 37 and N:P value 15; p=0.0033 between titers with N:P value 15 and N:P value 5.6).The data in Figure 22H were analyzed using one-way ANOVA and Tukey's multiple comparison test (redness diameters for N:P value 37 and N:P value 15 were p=0.0004; there was no significant difference between N:P value 15 and N:P value 5.6 or N:P value 3). A hypothetical model of the physical interaction between NLC and rvRNA, supported by these data, is illustrated in Figure 22D. [Figure 22-2] Same as above.

[0032] [Figure 23-1]Figures 23A–23E illustrate the characteristics of NLCv2 with enhanced RNA loading. Figure 23A shows particle size measured by DLS. Data are expressed as Z-mean. Figure 22B shows denatured RNA agarose gel electrophoresis of untreated rvRNA (lane 2), NLCv2 rvRNA (lane 4), ribonuclease-treated rvRNA (lane 3), and NLCv2 rvRNA (lane 5). Data are representative of three independent studies. Figure 23C shows the percentage (%) of RNA bound to NLCv1 or NLCv2, measured by densitometry analysis after denatured agarose gel electrophoresis of NLCv1 or NLCv2 complexed with gradually increasing concentrations of rvRNA. Data are representative of three independent experiments. Figure 23D shows SEAP expression in BHK cell supernatant after 24 hours incubation with NLCv1 or NLCv2 complexed with SEAP rvRNA at various N:P ratios. The data are presented as the mean ± standard deviation of three biological replicas and represent the results of three independent experiments. For comparison, NLCv1 data were replicated from Figure 23E. In Figure 23E, guinea pigs (n=4 / group) were inoculated via intramuscular or intradermal route with a single dose of 50 μg of unformulated ZIKV rvRNA, or 5 μg or 0.5 μg of NLCv1 formulated ZIKV rvRNA, or 50 μg, 5 μg, or 0.5 μg of NLCv2 formulated ZIKV rvRNA. Serum neutralizing antibody titers were quantified by PRNT80 measurement after 28 days. Each data point is listed with its mean ± standard deviation. The data were analyzed using Log10 transformation with one-way ANOVA and Tukey's multiple comparison test (comparing 5 μg doses of NLCv1 and NLCv2 formulations, with p=0.05 for intramuscular and p=0.04 for intradermal). [Figure 23-2] Same as above. [Figure 23-3] Same as above.

[0033] [Figure 24]Figures 24A to 24B. Figure 24A shows in vitro ZIKV neutralization by serial dilution of serum recovered in Figure 11C above. Each serum dilution was plotted as mean ± standard deviation, and a curve fitting was performed using an S-shaped nonlinear regression model. The data were analyzed using a two-way ANOVA and Tukey's multiple comparison test (at a serum dilution of 1 / 640, *p<0.05 for comparisons between NLC Span 60 and CNE Span 85, and between CNE Span 60 and CNE Span 85; **p<0.0001 for comparisons between NLC Span 60 and CNE Span 60). Figure 24B shows the Mip-1β concentrations measured by ELISA in the supernatant of human peripheral blood mononuclear cells (n=6 donors) recovered after 24 hours incubation with 40 ng of ZIKV rvRNA, either in its unformulated form (alone), or complexed with a CNE containing SPAN60 or SPAN85 in a squalene emulsion with an N:P value of 15, or complexed with an NLC containing Span60, Span80, or Span85 and squalene / Dynasan, or complexed with an NLC containing Span60 and Miglyol® / Dynasan emulsion. ELISA was performed on two technical replicates per donor. The mean values ​​for each donor are plotted along with the mean ± standard deviation of the six donors. The data were analyzed using a combined one-way ANOVA and Tukey's multiple comparison test (comparing NLC-Span60-squalene or NLC-Span85-squalene alone or with CNE-Span85, **p<0.005).

[0034] [Figure 25]Figure 25 shows the dynamics of protein expression after in vivo delivery of rvRNA formulated with NLCv1 or CNE. rvRNA encoding SEAP was formulated with NLCv1, CNE, or 10% sucrose (alone), and 100 ng was administered intramuscularly to C57BL / 6 mice (n=3 / group). Mock mice injected with 10% sucrose alone were used as negative controls. Blood was collected from mice on days 3, 7, 14, 21, and 28, and serum SEAP activity was measured by a SEAP assay.

[0035] [Figure 26] Figure 26 shows the optimization of the rvRNA load in NLCv1. The rvRNA encoding SEAP was diluted with 10% sucrose to final concentrations of 400 μg / mL, 300 μg / mL, 200 μg / mL, 100 μg / mL, and 40 μg / mL, and complexed with NLCv1 in a 1:1 ratio by gentle pipetting. CNE was complexed with 40 μg / mL rvRNA in a 1:1 ratio. Next, the complexed rvRNA or unformulated rvRNA was diluted with 10% sucrose for administration such that each 50 μL intramuscular injection yielded a 100 ng dose. After 3 days, blood was collected from the mice, and serum SEAP activity was measured by SEAP quantification and compared with mock-injected mice.

[0036] [Figure 27] Figure 27 shows the effect of reduced squalene concentration in high-volume NLC on serum SEAP activity. High-volume NLC (containing 3 w / v% DOTAP) prepared using 30 w / v%, 15 w / v%, 7.5 w / v%, and 3.75 w / v% squalene was complexed 1:1 with SEAP-encoding rvRNA at an N:P value of 37. 100 ng was administered intramuscularly to C57BL / 6 mice (n=3 / group), and serum SEAP activity was measured after 3 days and compared with unformulated rvRNA-injected mice or mock-injected mice.

[0037] [Figure 28-1]Figures 28A to 28G show the immunogenicity and efficacy of the NLCv1 / rvRNA complex in C57BL / 6 mice. NLCv1 was complexed with rvRNA or mRNA encoding ZIKV prM / E with an N:P value of 50, and a single dose of 1 μg was administered to C57BL / 6 mice by intramuscular injection (n=9 / group). Blood was collected 14 days later, and neutralizing antibody titers using PRNT80 were evaluated compared to mock-inoculated mice, or to 10 μg or 1 μg of rvRNA alone or mRNA alone (n=9 / group), or 1 μg of CNE-formulated rvRNA (N:P value 50) (Figure 28A). Data are shown as individual values ​​and as mean ± standard deviation. Thirty days after inoculation, six mice per group were challenged with the ZIKV Dakar 41525 strain in 5log10 PFU after antibody blockade of type I interferon as described in Smith et al., PLoS Negl Trop Dis 11(1):e0005296 (2017). Blood samples were collected four days later, and the amount of virus in the blood was quantified by plaque assay (Figure 20B). The mice were monitored daily for survival (Figure 29C) and weight loss (Figure 28D). The remaining three mice per group received a second inoculation on day 30, and the CD8+ T cell response after boosting was evaluated. After 14 days, the mice were euthanized, splenocytes were isolated and stained, and the percentage of B220loCD8+ T cells that were IFNγ-positive (Figure 28E), CD107a-positive (Figure 28F), or TNFα-positive (Figure 28G) was quantified by flow cytometry (Figure 37). [Figure 28-2] Same as above. [Figure 28-3] Same as above. [Figure 28-4] Same as above.

[0038] [Figure 29] Figure 29 illustrates an exemplary fusion protein ID91 (top panel) containing four Mtb antigens / proteins (Rv3619, Rv2389, Rv3478, and Rv1886), and the design of the alphavirus replicon encoding ID91. This replicon construct includes a Gaussia luciferase signaling molecule as a reporter gene.

[0039] [Figure 30-1] Figures 30A and 30B show the percentage (%) of CD4+ / CD44+ T cells (Figure 30A) or CD8+ / CD44+ T cells (Figure 30B) secreting TH1 cytokines from protein-based or RNA-based ID91 vaccines. Statistical significance (P<0.05) was analyzed using two-way ANOVA and is indicated by asterisks. [Figure 30-2] Same as above.

[0040] [Figure 31-1] Figures 31A–31D show data from proliferative or cytokine-producing CD4+ populations. Splenocytes derived from mice inoculated with ID91 protein / GLA-SE (blue) or ID91 RNA / QG807 (red) were stimulated with 13 peptide pools: culture medium, ID91 complete protein, and ID91 duplicate peptides pooled from 10 peptides. [Figure 31-2] Same as above.

[0041] [Figure 32-1] Figures 32A–32D show data from proliferative or cytokine-producing CD8+ populations. Splenocytes from mice inoculated with ID91 protein / GLA-SE (blue) or ID91 RNA / QG807 (red) were stimulated with 13 peptide pools: culture medium, ID91 complete protein, and ID91 duplicate peptides pooled from 10 peptides. [Figure 32-2] Same as above.

[0042] [Figure 33-1]Figures 33A and 33B show findings that inoculation with ID91 RNA provided prophylactic protection and resulted in differences in the induction of CD8+ T cell epitopes compared to ID91 protein. A mouse cohort was inoculated twice with ID91 protein + GLA-SE or alphaviral RNA encoding the ID91 antigen, 3 weeks apart. Splenocytes were isolated and restimulated in vitro with ID91 peptide. The intensity scale in the heatmap shows the percentage of CD4+ (red) and CD8+ (blue) T cell proliferation and cytokine response in response to each pool (Figure 30A). In Figure 33A, M represents medium only, ID91 represents ID91 alone, and PP1-PP13 represents peptide pools 1-13. The scale on the right shows the percentage of proliferative or cytokine-positive cells from the CD4+ or CD8+ population. Figure 33B shows the bacterial load assessed from lung homogenates 3 weeks after Mtb H37Rv challenge. A mouse cohort was inoculated once with either saline, ID91 protein + GLA-SE, or an RNA preparation containing alphaviral RNA encoding the ID91 antigen, followed by a second challenge three weeks later. Significance (p<0.05) compared to saline, as determined by one-way ANOVA and Dunnett's multiple comparison test, is indicated by asterisks. [Figure 33-2] Same as above.

[0043] [Figure 34-1]Figures 34A and 34B compare signal sequences derived from Zika virus (ZIKV) or Japanese encephalitis virus (JEV). Figure 34A shows the design of rvRNA encoding ZIKV prM / E, which has either a ZIKV signal sequence or a JEV signal sequence upstream of prM. In Figure 34B, ZIKV prM / E and rvRNA encoding either a ZIKV signal sequence or a JEV signal sequence were formulated in 10% sucrose (alone) or with a cationic nanoemulsion (CNE). 10 μg of the 10% sucrose formulation or 1 μg of the CNE formulation was injected intramuscularly into C57BL / 6 mice (n=5 / group), and serum PRNT80 titers were measured after 21 days. The mice were then boosted with a second inoculation, and PRNT80 titers were measured again after another 21 days. [Figure 34-2] Same as above.

[0044] [Figure 35] Figure 35 shows the effect of the surfactant-to-oil molar ratio on NLC particle size (Z-mean (nm)). The surfactant-to-oil ratio was changed by varying the total amount of surfactant while keeping the oil content constant. The experimental data were approximated by a single-phase exponential decay equation (R² = 0.972). All formulations were treated with a microfluidizer under the same conditions (10 cycles at 30,000 psi).

[0045] [Figure 36-1]Figures 36A and 36B show the effects of hydrophilic surfactants on protection from ribonuclease challenge and delivery of SEAP-expressing rvRNA in vivo. rvRNA was complexed with various NLCs containing 70% or 35% total surfactant and cationic lipids, prepared with or without 10 mM citrate buffer, and its ability to protect rvRNA from ribonuclease challenge was evaluated (Figure 36A). C57BL / 6 mice (n=3 / group) were administered rvRNA encoding SEAP complexed with 10% sucrose (alone), or NLCs containing 70% or 35% total surfactant and cationic lipids, or CNEs. Serum SEAP activity was measured after 3 days (Figure 36B). [Figure 36-2] Same as above.

[0046] [Figure 37] Figure 37 shows an exemplary flow cytometry gating strategy.

[0047] [Figure 38] Figures 38A and 38B illustrate examples of ID91 modified with restriction enzymes. Figure 38A shows the pET29 vector, and Figure 38B shows the pET28 vector.

[0048] [Figure 39-1]Figures 39A to 39E show innate immune signaling in human PBMC-derived dendritic cells (DCs) after stimulation with TLR3 (Roboxxol, pIC:HMW) agonists and RIG-I (SEVDI) agonists, with or without NLC formulations. PBMC-DCs obtained from six human donors were stimulated with either NLC-formulated polyIC:HMW ("QG942 formulation") or polyIC:HMW, Riboxxol, or SEVDI alone ("unformulated"). The formulation-only control was labeled "Culture control." After incubation at 37°C and 5% CO2 for 24 hours, the supernatant was quantified for innate immune marker concentrations using a commercially available ELISA kit. Statistical analysis was performed using two-way ANOVA and Sidaq's multiple comparison test. P-values: *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001. [Figure 39-2] Same as above. [Figure 39-3] Same as above.

[0049] [Figure 40] Figure 40 shows the induction of IFNα / β in MM6 cells stimulated with Riboxxol adjuvant or pIC:HMW adjuvant. Cells were stimulated with RNA alone or formulated RNA. The formulations are shown in Table XX. IFNα / β was measured from the supernatant of stimulated MM6 cells compared to unstimulated MM6 cells and determined using the HEK-blue IFNα / β SEAP reporter cell line. The dotted line represents relative stimulation, measured after the addition of known (nown) IFNα concentrations.

[0050] [Figure 41] Figure 41 shows a heatmap summarizing IFNα / β induction as a function of N:P molar ratio and riboxxol dose.

[0051] [Figure 42-1] Figures 42A to 42D show cytokine expression after administration of NLC or SE and formulated dsRNA adjuvants. [Figure 42-2] Same as above. [Figure 42-3] Same as above.

[0052] [Figure 43] Figure 43 shows the decrease in SEAP expression after administration of dsRNA adjuvant (TLR3 ligand) and formulated SEAP with NLC.

[0053] [Figure 44] Figures 44A and 44B show IP-10 (Figure 42A) and SEAP expression (Figure 42B) after administration of formulated VEErep-SEAP to NLC, SE, or control. In Figure 42A, *** = statistical significance of the comparison between 1 μg + QG942 and 1 μg alone. In Figure 42B, * = statistical significance of the comparison between 1 μg + QG942 and 0.1 μg + QG942, ** = statistical significance of the comparison between 0.1 μg + QG942 and 0.1 μg SE or alone, and *** = statistical significance of the comparison between 0.1 μg + QG942 and 0.1 μg alone. [Figure 45-1] Figure 45 shows the antigen-specific antibody response. C57BL / 6 mice were injected with 1 μg of LEISH-F2 protein mixed with the indicated adjuvant formulation twice, 3 weeks apart. Serum was collected on day 21 (pre-boost) and day 42, and antigen-specific endpoint titers of IgG, IgG1, and IgG2c were measured by ELISA. Data are shown as mean + SD for 5 mice / group. [Figure 45-2] Same as above.

[0054] [Figure 46]Figures 46A and 46B show the effect of Hiltonol® formulations on T cell responses. C57BL / 6 mice were injected with 1 μg of LEISH-F2 protein mixed with the indicated adjuvant formulation twice, 3 weeks apart. Three weeks after the last inoculation, the spleen was removed to prepare single-cell suspensions, and the cells were incubated with LEISH-F2 antigen. The cytokine levels in the culture supernatant were then measured by ELISA. Data are shown as minimum and maximum values, boxes indicating the 25th and 75th percentiles, and the mean values ​​indicated by horizontal bars within the boxes (5 mice / group).

[0055] [Figure 47-1] Figures 47A–47E show the effect of Hiltonol® formulations on T cell responses. C57BL / 6 mice were injected with 1 μg of LEISH-F2 protein mixed with the indicated adjuvant formulation twice, 3 weeks apart. Three weeks after the last inoculation, the spleen was removed to prepare a single-cell suspension, and the cells were incubated with the F2 antigen. The phenotype of the cells was then determined by flow cytometry. Data are presented as individual points for each mouse, as well as the mean value + SEM, indicated by horizontal and vertical bars, respectively. N=5 mice / group. [Figure 47-2] Same as above. [Figure 47-3] Same as above.

[0056] [Figure 48] Figure 48 shows the effect of Hiltonol® formulations on antigen-specific antibody responses. C57BL / 6 mice were injected once with 1 mg of LEISH-F3+ protein mixed with the indicated adjuvant formulation. Four weeks after inoculation, serum was collected and antigen-specific IgG endpoint titers were measured by ELISA. Data are shown as individual points for each animal, as well as mean values ​​+ SEM. N=5 mice / group.

[0057] [Figure 49-1]Figures 49A–49C show the effect of Hiltonol® formulations on T cell responses. C57BL / 6 mice were injected once with 1 μg of LEISH-F3+ protein mixed with the indicated adjuvant formulation. Four weeks after the last inoculation, the spleens were removed to prepare single-cell suspensions, and the cells were incubated with F3+ protein or MHCI restriction peptide (CD8 T cell epitope). Cytokine levels in the culture supernatant were then measured by ELISA. Data are presented as individual points for each mouse, as well as mean values ​​+ SEM, indicated by horizontal and vertical bars, respectively. N=5 mice / group. [Figure 49-2] Same as above.

[0058] [Figure 50-1] Figures 50A to 50G show the effects of antigen-vaccine delivery on immunogenic responses with various formulations. [Figure 50-2] Same as above.

[0059] [Figure 51] Figures 51A and 51B show the effects of antigen-vaccine delivery on immunogenic responses with various formulations.

[0060] [Figure 52-1] Figures 52A to 52C show the effects of antigen-vaccine delivery on immunogenic responses with various formulations. [Figure 52-2] Same as above. [Figure 52-3] Same as above.

[0061] Table 1 lists the specific sequences referenced in this specification. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Modes for carrying out the invention]

[0062] This specification provides compositions for delivering bioactive agents to cells, and methods for such delivery. NLCs have a core composed of a combination of liquid-phase lipids and solid-phase lipids. Unlike solid lipid nanoparticles (LNPs) which have a completely solid crystalline core, the mixed-phase core of NLCs offers greater flexibility and can incorporate active molecules of various structures. While not limited to theory, it is believed that by adding solid lipids to this composition, the NLC core gains structural integrity and stability. In the NLCs of this invention, the oily core of the mixed phase is emulsified with a mixture of surfactants (typically sorbitan esters and hydrophilic surfactants) and cationic components (typically cationic lipids or phospholipids). Typically, bioactive agents such as small molecule drugs are incorporated into the oily core of the NLC, but the inventors have synthesized an NLC (i.e., the bioactive agent is not encapsulated by the NLC) that can interact on or near the surface with bioactive agents such as negatively charged molecules (e.g., RNA). The inventors have found that the NLC is stable, capable of delivering nucleic acids to cells, and that the nucleic acid, which is a bioactive agent, and the NLC can be manufactured and stored separately and mixed immediately before use. Therefore, the NLC can be used in a variety of applications, including a fast-response nucleic acid platform technology for developing multiple preventive or therapeutic treatments. This fast-response nucleic acid delivery platform is based on a flexible system that utilizes the NLC composition to deliver nucleic acids (e.g., replicating viral RNA (rvRNA)) that drive RNA replication and / or protein expression (e.g., resulting in a robust and rapid immune response to a wide variety of viral, bacterial, or parasitic antigens). The NLC composition of the present invention can be stored for a long period after manufacture. Subsequently, the stored base can be combined with nucleic acids (e.g., synthetic rvRNA expressing infection-protective antigens) in the event of an emerging disease outbreak or other public health event.

[0063] In addition to providing NLCs for combination with bioactive agents, NLCs that are already combined are also provided. That is, in some embodiments, the bioactive agent is bound to the NLC. By administering this NLC-bioactive agent composition, the bioactive agent can be delivered to the target when needed.

[0064] I. Definition

[0065] Unless otherwise specified, the following terms have the meanings set forth below. Any terms for which no definition is provided have the meaning recognized in the relevant technical field.

[0066] In this specification, unless otherwise specified, the terms “about” and “consisting essentially of” mean ±20% of the range, value, or structure indicated.

[0067] The use of alternatives (e.g., "or") should be understood to mean one of the alternatives, both of them, or any combination thereof.

[0068] As used herein, the terms “include,” “have,” and “comprise” are used synonymously, and these terms and their variations are intended to be interpreted as non-restrictive.

[0069] As used herein and in the appended claims, the singular forms "a," "an," and "the" include the plural form unless otherwise explicitly indicated by the context.

[0070] As used herein, the term "macromolecule" refers to large molecules, including, but not limited to, peptides, proteins, oligonucleotides, and polynucleotides, whether of biological or synthetic origin.

[0071] The term "alkyl" refers to aliphatic hydrocarbons that are linear or branched, acyclic or cyclic, unsaturated or saturated, and contain the indicated number of carbon atoms. Unsaturated alkyls contain at least one double or triple bond between adjacent carbon atoms.

[0072] The terms “polypeptide,” “peptide,” and “protein” are used synonymously herein and refer to amino acid polymers of any length. These polymers may be linear or branched, may contain modified nucleotides or modified amino acids, and may be further interrupted by non-nucleotides or non-amino acids. These terms also encompass naturally modified or intervened nucleotide polymers or amino acid polymers, such as those modified by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation or modification, such as binding with a labeling component. Polynucleotides or polypeptides containing one or more nucleotide analogs or amino acid analogs (e.g., non-natural amino acids) are also included in the above definitions and may contain other modifications known in the art.

[0073] The term "isolated" means that the molecule has been removed from its natural environment.

[0074] "Purified" means that the purity of a molecule has been increased so that it exists in a purer form compared to when it is present in its natural environment and / or when it was first synthesized and / or amplified under laboratory conditions. Purity is a relative term and does not necessarily mean absolute purity.

[0075] "Polynucleotide" or "nucleic acid" are used synonymously herein and refer to nucleotide polymers of any length, including DNA and RNA. Such nucleotides may be, for example, deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substrate that can be incorporated into the polymer by DNA polymerase or RNA polymerase or by synthetic reaction. Polynucleotides may include modified nucleotides such as methylated nucleotides and their analogs. If modifications are made, the modification of the nucleotide structure may be performed before or after polymer construction.

[0076] As used herein, “oligonucleotide” typically refers to a short, usually single-stranded, usually synthetic, but not necessarily, polynucleotide, typically less than approximately 200 nucleotides in length. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The above descriptions concerning polynucleotides apply equally and completely to oligonucleotides.

[0077] The term "individual" or "subject" is not particularly limited to mammals. Examples of mammals include, but are not limited to, humans, primates, livestock, sport animals, pets (cats, dogs, horses), and rodents.

[0078] As used herein, "replicon" includes any genetic element that enables mass replication under its own control, such as plasmids, cosmids, bacmids, phages, or viruses. Replicons may be RNA or DNA, and may be single-stranded or double-stranded.

[0079] Vitamin E refers to both tocopherol (TCP) and tocotrienol, and can be considered either natural or synthetic vitamin E.

[0080] Monoacylglycerol is an ester of glycerol, a trihydric alcohol, in which one of the hydroxyl groups is esterified with a long-chain fatty acid.

[0081] Lauroyl polyoxylglycerides are mixtures of glycerol monoesters, diesters, and triesters, as well as polyethylene glycol monoesters and diesters, with a typical average relative molecular weight of approximately 300 to 1500.

[0082] Tri(caprylic / capric)glyceride is a mixture of glycerin triester, caprylic acid, and capric acid.

[0083] The term liquid-phase lipids refers to lipids that are liquid at room temperature before being mixed with other components.

[0084] The term "solid-phase lipid" refers to lipids that are solid at room temperature before being mixed with other components.

[0085] The room temperature is between 15°C and 25°C.

[0086] Glycerolipids are fatty molecules composed of glycerol esterified with fatty acids. Glycerolipids include triglycerides and diglycerides.

[0087] The term "sorbitan ester," as used herein, refers to an ester of sorbitan. Sorbitan is represented by formula A.

[0088] [ka] Formula A

[0089] Particularly preferred sorbitan esters are those with an alkyl group of C1-C 30 Alkyl alkyl groups, preferably saturated or unsaturated C1-C 20 Alkyl alkyl groups, more preferably saturated or unsaturated C 10 ~C 20It is an alkyl group, specifically a sorbitan alkyl ester.

[0090] Unless otherwise specified, the implementation of this invention will utilize conventional molecular biology, recombinant DNA, biochemistry, and chemistry techniques within the scope of the skills of those skilled in the art. Such techniques are adequately described in the following literature. For example, Molecular Cloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed., Cold Spring Harbor Laboratory Press: (1989); DNA Cloning, Volumes I and II (DN Glover ed., 1985); Oligonucleotide Synthesis (MJ Gait ed., 1984); Mullis et al., US Pat. No: 4,683,195; Nucleic Acid Hybridization (BD Hames & SJ Higgins eds. 1984); B. Perbal, A Practical Guide to Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., NY); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989).

[0091] II. Nanostructured Lipid Carriers

[0092] This disclosure provides, in particular, NLCs for delivering bioactive agents to cells. The NLC composition comprises NLC particles comprising (a) an oily core composed of liquid-phase lipids and solid-phase lipids, (b) a cationic component (preferably a cationic lipid or phospholipid), (c) a hydrophobic surfactant, preferably a sorbitan ester (e.g., sorbitan monoester, sorbitan diester, or sorbitan triester), and (d) a surfactant (preferably a hydrophilic surfactant). Typically, the NLC of the present invention comprises an unstructured, i.e., amorphous, solid lipid matrix composed of a mixture of solid lipids and liquid lipids dispersed in an aqueous phase. One or more of the surfactants described above may be present in the oil phase, the aqueous phase, or at the interface between the oil phase and the aqueous phase. In some embodiments, the sorbitan ester and the cationic lipid are present at the interface between the oil phase and the aqueous phase.

[0093] The inventors have found that the claimed NLC is particularly effective in the delivery of protein-coding nucleic acids (such as RNA). Furthermore, they have found that by manipulating specific components of the NLC, the expression level of the encoded protein can be increased. Surprisingly, the exemplary NLC can not only effectively deliver RNA but also enhance the immune response to the encoded protein.

[0094] A. Solid phase lipids and liquid phase lipids

[0095] NLC is composed of a mixture of solid and liquid lipids. The liquid and solid lipids used in NLC are not particularly limited, as long as they are lipids capable of forming an unstructured or amorphous solid lipid matrix and creating a stable composition. The weight ratio of solid to liquid can vary widely, for example, from 0.1:99.9 to 99.9:0.1. In some exemplary embodiments, solid lipids are mixed with liquid lipids at a solid lipid:liquid lipid weight ratio of about 70:30 to about 99.9:0.1 or about 1:10 to about 1:30. In some embodiments, solid lipids are mixed with liquid lipids at a solid lipid:liquid lipid weight ratio of about 1:16.

[0096] The total oily core components (solid lipids + liquid oil) of an NLC-based composition or formulation are typically present in amounts of about 0.2% to about 50% (w / v). For example, the NLC may contain about 0.2% to about 50% (w / v) of oily core components, 0.2% to about 40% (w / v) of oily core components, about 0.2% to about 30% (w / v) of oily core components, about 0.2% to about 20% (w / v) of oily core components, about 0.2% to about 15% (w / v) of oily core components, about 0.2% to about 10% (w / v) of oily core components, about 0.2% to about 9% (w / v) of oily core components, and about 0. Oily core components of 2% to approximately 8% (w / v), oily core components of approximately 0.2% to approximately 7% (w / v), oily core components of approximately 0.2% to approximately 6% (w / v), oily core components of approximately 0.2% to approximately 5% (w / v), oily core components of approximately 0.2% to approximately 4.3% (w / v), oily core components of approximately 0.3% to approximately 20% (w / v), oily core components of approximately 0.4% to approximately 20% (w / v), oily core components of approximately 0.5% to approximately 20% (w / v) A component, approximately 1% to approximately 20% (w / v) oily core component, approximately 2% to approximately 20% (w / v) oily core component, approximately 3% to approximately 20% (w / v) oily core component, approximately 4% to approximately 20% (w / v) oily core component, approximately 5% to approximately 20% (w / v) oily core component, approximately 0.5% (w / v) oily core component, approximately 1% (w / v) oily core component, approximately 1.5% (w / v) oily core component, approximately 2% (w / v) oily core The formulation may contain approximately 2.5% (w / v) of an oily core component, approximately 3% (w / v) of an oily core component, approximately 3.5% (w / v) of an oily core component, approximately 4% (w / v) of an oily core component, approximately 4.3% (w / v) of an oily core component, approximately 5% (w / v) of an oily core component, or approximately 10% (w / v) of an oily core component, and any other content or range of the oily core component as described herein. In particular, when considering diluted or concentrated formulations, higher w / v% or lower w / v% are intended herein.

[0097] The oily core of the NLC contains liquid-phase lipids. While not necessarily required, the liquid-phase lipids are preferably metabolizable, non-toxic oils, more preferably oils with approximately 6 to 30 carbon atoms, including, but not limited to, alkanes, alkenes, alkynes, and their corresponding acids and alcohols, their ethers and esters, and mixtures thereof. The oils may be any vegetable oil, fish oil, animal oil, or synthetically produced oil that can be administered to a target. In some embodiments, the liquid-phase lipids are non-metabolizable lipids.

[0098] The oil may be, for example, any long-chain alkane, long-chain alkene or long-chain alkyne, or an acid derivative or alcohol derivative such as a free acid, its salt, or an ester such as a monoester, diester or triester, for example, triglycerides and esters of 1,2-propanediol or similar polyhydric alcohols. The alcohol may be acylated, and monofunctional or polyfunctional acids, such as acetic acid, propanoic acid, or citric acid, may be used. Long-chain alcohol-derived ethers that are oils and meet other criteria described herein may also be used.

[0099] Each alkane, alkene, or alkyne moiety, and its acid or alcohol derivative, typically has about 6 to about 40 or 6 to about 30 carbon atoms. The moiety may have a linear or branched structure. The moiety may be fully saturated or have one or more double or triple bonds. When using monoester, polyester, or ether-based oils, the limitation of about 6 to about 40 carbon atoms applies to each individual fatty acid or aliphatic alcohol moiety, not to the total number of carbon atoms.

[0100] Any oil derived from animals, fish, or vegetables may be used, if suitable. Sources of vegetable oils include nuts, seeds, and grains, with suitable oils including, for example, peanut oil, soybean oil, coconut oil, and olive oil. Other suitable seed oils include safflower oil, cottonseed oil, sunflower seed oil, and sesame oil. Among grains, corn oil, as well as oils from the grains of other cereals such as wheat, oats, rye, rice, teff, and rye, may be used. Techniques for obtaining vegetable oils are well-established and well-known. Compositions of these oils and other similar oils can be found, for example, in the Merck Index and in materials relating to food, nutrition, and food technology.

[0101] Most fish contain readily recoverable metabolizable oils. Examples of fish oils usable herein include, for example, cod liver oil, shark liver oil, and whale oil such as whale wax. Several branched-chain oils have been biochemically synthesized using 5-carbon isoprene units, and these are generally called terpenoids. Natural or synthetic terpenoids, also known as isoprenoids, can be used herein as liquid-phase lipids. In this specification, squalene, a branched-chain unsaturated terpenoid, is particularly preferred. Shark liver oil is the primary source of squalene, but vegetable oils (mainly vegetable oils), including amaranth seed, rice bran, wheat germ, and olive oil, are also suitable sources. Squalane, a saturated analog of squalene, is also preferred. Oils, including fish oils such as squalene and squalane, can be readily obtained from commercial sources and may be acquired by methods known in the art. Oils used herein may also be produced using synthetic means, including genetic engineering (e.g., oil produced from genetically modified yeast containing squalene).

[0102] Exemplary liquid-phase lipids that can be used in the present invention include, for example, castor oil, coconut oil, corn oil, cottonseed oil, evening primrose oil, fish oil, grape seed oil, jojoba oil, lard oil, flaxseed oil, olive oil, peanut oil, safflower oil, sesame oil, soybean oil, squalene, squalane, sunflower oil, wheat germ oil, mineral oil, caprylic / capric triglyceride (e.g., Myglyol® 810, Myglyol® 812, Labrafac®), vitamin E (e.g., TOS, TPGS), lauroyl polyoxylglycerides (e.g., Gelucire® 44 / 14), monoacylglycerols (e.g., Myverol 18-99 K), soybean lecithin (e.g., Epikuron® 200), farnesene, or combinations thereof.

[0103] The liquid-phase lipids may include, for example, squalene, sunflower oil, soybean oil, olive oil, grape seed oil, squalane, caprylic / capric triglyceride, or combinations thereof.

[0104] The liquid-phase lipid may include, for example, squalene, squalene tri(caprylic / capric acid)glyceride, or a combination thereof.

[0105] The liquid-phase lipid may include, for example, tri(caprylic / capric acid) glyceryl, vitamin E, lauroyl polyoxylglyceride, monoacylglycerol, soy lecithin, squalene, or squalane, or a combination thereof.

[0106] The liquid-phase lipid may include, for example, squalene, farnesene, or a combination thereof.

[0107] The oily core of the NLC contains solid-phase lipids. Various solid-phase lipids can be used, for example, glycerolipids. Exemplary solid-phase lipids include, for example, glyceryl lumitostearate (Precitol ATO® 5), glyceryl monostearate, glyceryl dibehenate (Compritol® 888 ATO), cetyl palmitate (Crodamol® CP), stearic acid, tripalmitin, or microcrystalline triglycerides. Exemplary microcrystalline triglycerides include those sold under the trade name Dynasan® (for example, trimiristin (Dynasan® 114), tristearin (Dynasan® 118), or tripalmitin (Dynasan® 116)).

[0108] The solid-phase lipid can be, for example, a microcrystalline triglyceride, such as one selected from trimiristin (Dynasan® 114) or tristearin (Dynasan® 118).

[0109] The solid lipid phase of the oily core is preferably solid at room temperature. Indoors, room temperature is typically 15°C to 25°C.

[0110] In any of the embodiments provided herein, the solid-phase lipid may be a glycerolipid, such as a microcrystalline triglyceride.

[0111] In any of the embodiments provided herein, the liquid-phase lipid may be synthetic squalene or natural squalene.

[0112] B. Cationic components

[0113] The NLC described herein comprises a cationic component, typically a cationic lipid. The cationic component is useful in its interaction with negatively charged bioactive agents on the surface of the NLC. Any cationic lipid can be used as long as it does not impair the stability of the NLC, can be administered to a subject, and is capable of interacting with negatively charged bioactive agents. Typically, the cationic lipid contains nitrogen atoms that are positively charged under physiological conditions. Suitable cationic lipids include: benzalkonium chloride (BAK), benzethonium chloride, cetrimide (containing tetradecyltrimethylammonium bromide, and possibly containing small amounts of dodecyltrimethylammonium bromide and hexadecyltrimethylammonium bromide), cetylpyridinium chloride (CPC), cetyltrimethylammonium chloride (CTAC), primary amines, secondary amines, tertiary amines, for example, but not limited to N,N′,N′-polyoxyethylene(10)-N-tallow-1,3-diaminopropane, other quaternary amine salts, for example, but not limited to dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, mixed alkyl-trimethylammonium bromide, benzyldimethyldodecylammonium chloride, benzyldimethylhexadecylammonium chloride, benzyltrimethylammonium Nium methoxide, cetyldimethylethylammonium bromide, dimethyldioctadecylammonium bromide (DDAB), methylbenzethonium chloride, decamethonium chloride, methyl mixed trialkylammonium chloride, methyltrioctylammonium chloride, N,N-dimethyl-N-[2(2-methyl-4-(1,1,3,3-tetramethylbutyl)-phenoxy]-ethoxy)ethyl]benzenemethaneaminium chloride (DEBDA), dialkyldimethylammonium salt, [1-(2,3-dioleyloxy)-propyl]-N,N,N,trimethylammonium chloride, 1,2-diacyl-3-(trimethylammonio)propane (acyl group = dimyristoyl, dipalmitoyl, distearoyl, dioleoyl), 1,2-diacyl-3(dimethylammonio)propane (acyl group = dimyristoyl, dipalmitoyl, distearoyl, dioleoyl), 1,2-Dioleoyl-3-(4′-trimethylammonio)butanoyl-sn-glycerol, 1,2-Dioleoyl-3-succinyl-sn-glycerolcholine ester, cholesteryl(4′-trimethylammonio)butanoate), N-alkylpyridinium salts (e.g., cetylpyridinium bromide and cetylpyridinium chloride), N-alkylpiperidinium salts, dicationic poro-type electrolytes (C12Me6;C12Bu6), dialkylglycetylphosphorylcholine, lysolecithin, L-α-dioleo Ilphosphatidylethanolamine, cholesterol hemisuccinate choline ester, lipopolyamine, for example, but not limited to dioctadecylamideglycylspermine (DOGS), dipalmitoylphosphatidylethanol-amidespermine (DPPES), lipopoly-L(or D)-lysine (LPLL, LPDL), N-glutarylphosphatidylethanolamine-linked poly(L(or D)-lysine), didodecyl glutamate ester (C1) having an amino group as a pendant group. Ditetradecyl glutamate esters (C14GluCnN+) having an amino group as a pendant group, cationic derivatives of cholesterol, for example, but not limited to cholesteryl-3β-oxysuccinamide ethylenetrimethylammonium salt, cholesteryl-3β-oxysuccinamide ethylenedimethylamine, cholesteryl-3β-carboxyamide ethylenetrimethylammonium salt, cholesteryl-3β-carboxyamide ethylenedimethylamine, and 3γ-[N-(N′,N-dimethylaminoethanecarbomoyl]cholesterol] (DC-cholesterol), 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), dimethyldioctadecylammonium (DDA), 1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP), dipalmitoyl(C16:0)trimethylammoniumpropane (DPTAP), distearoyltrimethylammoniumpropane (DSTAP), and combinations thereof.

[0114] Other cationic lipids suitable for use in the present invention include, for example, the cationic lipids described in U.S. Patent Publication No. 2008 / 0085870 (published on April 10, 2008) and U.S. Patent Publication No. 2008 / 0057080 (published on March 6, 2008).

[0115] Other cationic lipids suitable for use in the present invention include, for example, the lipids E0001-E0118 or E0119-E0180 as listed in Table 6 (pages 112-139) of International Publication No. 2011 / 076807 (the said International Publication also describes methods for preparing and using these cationic lipids). Additional suitable cationic lipids include N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), and 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA).

[0116] This NLC may contain one of the cationic lipids described herein, or any combination of two or more of them.

[0117] In exemplary embodiments, the cationic lipid is selected from the group consisting of 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 313-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol), dimethyldioctadecylammonium (DDA), 1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP), dipalmitoyl(C16:0)trimethylammoniumpropane (DPTAP), distearoyltrimethylammoniumpropane (DSTAP), lipids E0001-E0118 or E0119-E0180 as listed in Table 6 (pages 112-139) of International Publication No. 2011 / 076807, and combinations thereof.

[0118] In other exemplary embodiments, the cationic lipid is 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 313-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol), dimethyldioctadecylammonium (DDA), 1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP), dipalmitoyl(C16:0)trimethylammoniumpropane (DPTAP), distearoyltrimethylammoniumpropane (DSTAP), N-[1-(2,3-dioleyloxy)propyl] The lipids selected are those listed in Table 6 (pages 112-139) of International Publication No. 2011 / 076807, including lipids E0001-E0118 or E0119-E0180, and combinations thereof.

[0119] Exemplary cationic lipids are selected from the following: 1,2-Dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol), dimethyldioctadecylammonium (DDA), 1,2-Dimyristoyl-3-trimethylammoniumpropane (DMTAP), dipalmitoyl (C 16:0) Trimethylammonium propane (DPTAP), distearoyltrimethylammonium propane (DSTAP), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), or combinations thereof. Further preferred cationic lipids may be those known to those skilled in the art.

[0120] In some embodiments, the NLC-based composition or formulation contains about 0.5 mg / mL to about 50 mg / mL of the cationic component (e.g., the cationic lipid). In some embodiments, the cationic lipid is DOTAP. The NLC may contain, for example, about 0.5 mg / mL to about 25 mg / mL or 30 mg / mL of DOTAP, or it may contain any other content or range of DOTAP as described herein.

[0121] In one embodiment, the cationic lipid is DC cholesterol. In one embodiment, the NLC may contain about 0.1 mg / mL to about 5 mg / mL of DC cholesterol. In one embodiment, the cationic lipid is DDA. The NLC may contain, for example, about 0.1 mg / mL to about 5 mg / mL of DDA. In one embodiment, the cationic lipid is DOTMA. The NLC may contain, for example, about 0.5 mg / mL to about 25 or 30 mg / mL of DOTMA. In one embodiment, the cationic lipid is DOEPC. The NLC may contain, for example, about 0.5 mg / mL to about 25 mg / mL of DOEPC. In one embodiment, the cationic lipid is DSTAP. The NLC may contain, for example, about 0.5 mg / mL to about 50 mg / mL of DSTAP. In one embodiment, the cationic lipid is DODAC. This NLC may contain, for example, about 0.5 mg / mL to about 50 mg / mL of DODAC. In one embodiment, the cationic lipid is DODAP. This NLC may contain, for example, about 0.5 mg / mL to about 50 mg / mL of DODAP.

[0122] In w / v terms, an example NLC-based composition or formulation contains, for example, about 0.05 w / v% to about 5 w / v% or about 10 w / v% of cationic components (e.g., cationic lipids such as DOTAP), about 0.2 w / v% to about 10 w / v% of cationic components (e.g., cationic lipids such as DOTAP), about 0.2 w / v% to about 5 w / v% of cationic components (e.g., cationic lipids such as DOTAP), about 0.2 w / v% to about 2 w / v% of cationic components (e.g., cationic lipids such as DOTAP), about 2 w / v% to 10 w / v% of cationic components (e.g., The formulation may contain cationic lipids such as DOTAP, approximately 2 w / v% to approximately 5 w / v% of cationic components (e.g., cationic lipids such as DOTAP), approximately 1 w / v% to approximately 5 w / v% of cationic components (e.g., cationic lipids such as DOTAP), approximately 3 w / v% to approximately 5 w / v% of cationic components (e.g., cationic lipids such as DOTAP), or approximately 3 w / v% to approximately 4 w / v% of cationic components (e.g., cationic lipids such as DOTAP), or any other content or range of cationic components as described herein with respect to cationic components. In particular, when considering diluted or concentrated formulations, higher w / v% or lower w / v% are intended herein.

[0123] In some cases, it may be preferable to use a soluble cationic lipid in the oily core. For example, DOTAP, DOEPC, DODAC, and DOTMA are soluble in squalene or squalane. In other cases, it may be preferable to use an insoluble cationic lipid in the oily core. For example, DDA and DSTAP are insoluble in squalene. Determining whether a particular lipid is soluble or insoluble in the oil and selecting an appropriate oil-lipid combination accordingly is within the scope of the knowledge of the art. For example, solubility can be predicted from the structure of the lipid and oil (for example, the solubility of a lipid can be determined by the structure of its terminals). For example, lipids having one or two unsaturated fatty acid chains (e.g., oleoyl terminals), such as DOTAP, DOEPC, DODAC, and DOTMA, are soluble in squalene or squalane; lipids having saturated fatty acid chains (e.g., stearoyl terminals) are insoluble in squalene. Alternatively, solubility can be determined according to the amount of lipid that dissolves in a given amount of oil and forms a saturated solution.

[0124] The NLC may contain additional lipids (i.e., neutral and anionic lipids) in combination with the cationic lipid, provided that the net surface charge of the NLC before mixing with the bioactive agent is positive. Methods for measuring the surface charge of the NLC are known in the art and include, for example, measurements by dynamic light scattering (DLS), photon correlation spectroscopy (PCS), or gel electrophoresis.

[0125] C. Sorbitan monoester

[0126] The inventors have found that when a bioactive agent is an antigen or encodes an antigen, and the composition is administered to a subject, sorbitan esters added to the NLC can enhance the effectiveness of the NLC in delivering the bioactive agent to cells and / or in inducing antibodies against the antigen in the subject. In particular, it was found that the immune response to proteins encoded by bioactive nucleic acids can be modulated by the selection of the sorbitan ester used in the NLC. A surprising finding was obtained that the use of sorbitan monoester is particularly effective in enhancing the effectiveness of the NLC. In some embodiments, the acyl chain of the sorbitan monoester is saturated. Furthermore, although not limited to theory, it was surprising found that the sorbitan ester, in particular sorbitan monoester, in combination with solid lipids (e.g., microcrystalline triglycerides), enhances the effectiveness of the NLC's adjuvant activity (e.g., effectiveness in inducing antibodies against the antigen in a subject when a bioactive agent is an antigen or encodes an antigen, and the composition is administered to a subject).

[0127] Exemplary sorbitan monoesters are commercially available under the trade names SPAN® or ARLACEL®. Exemplary sorbitan monoesters as used herein may be represented as the compound of formula I or its stereoisomers (e.g., formulas Ia, Ib, Ic, or Id, but not limited to these), where R is a saturated or unsaturated C1-C153 compound. 30 Alkyl alkyl groups, preferably saturated or unsaturated C1-C 20 Alkyl alkyl groups, more preferably saturated or unsaturated C 10 ~C 20 It is an alkyl group. In exemplary embodiments, the alkyl group is an acyclic alkyl group. Exemplary sorbitan monoesters also include positional isomers of formulas I, Ia, Ib, Ic or Id (for example, one of the hydroxyl functional groups is an ester functional group (for example, the alkyl is saturated or unsaturated C1-C) 30 Alkyl alkyl groups, preferably saturated or unsaturated C1-C 20 Alkyl alkyl groups, more preferably saturated or unsaturated C10 ~C 20 an alkyl group, and where R is OH, is substituted with an alkyl ester). It will be understood by those skilled in the art that exemplary sorbitan monoesters may also be in the form of salts of formula I, formula Ia, formula Ib, formula Ic, formula Id, and their stereoisomers or positional isomers (e.g., pharmaceutically acceptable salts). [Chemical formula]

[0128] In this regard, particularly preferred sorbitan monoesters are sorbitan monostearate (also known as Span® 60, shown below) and sorbitan monooleate (also known as Span® 80, shown below), although other sorbitan monoesters can also be used (e.g., but not limited to, sorbitan monolaurate (Span® 20), sorbitan monopalmitate (Span® 40)). Exemplary sorbitan monostearate is represented by formula II or formula IIa or a salt form thereof, and exemplary sorbitan monooleate is represented by formula III or formula IIIa or a salt form thereof. [Chemical formula]

[0129] In addition to using sorbitan monoester as a component of NLC, it is also contemplated to replace sorbitan monoester with another hydrophobic surfactant. For example, replacement with another sorbitan-based nonionic surfactant can be mentioned. That is, in this specification, oil cores composed of liquid-phase lipids and solid-phase lipids, cationic components (preferably cationic lipids or phospholipids), hydrophobic surfactants (for example, nonionic surfactants including sorbitan-based nonionic surfactants), and hydrophilic surfactants are also provided. NLC particles. Examples of sorbitan-based nonionic surfactants include sorbitan esters other than sorbitan monoester, such as sorbitan diester and sorbitan triester, such as sorbitan trioleate (SPAN85 (trademark)) and sorbitan tristearate (SPAN65 (trademark)). Usually, the nonionic surfactant (including sorbitan-based nonionic surfactant) will have a hydrophilic-lipophilic balance (HLB) value of 1.8 to 8.6. All embodiments provided in this specification of NLC containing sorbitan monoester are applicable to NLC containing another hydrophobic surfactant instead of sorbitan monoester, for example, NLC containing sorbitan diester or sorbitan triester instead of sorbitan monoester, and are contemplated. Sorbitan diester and sorbitan triester or other hydrophobic surfactants can be present at the same concentration as sorbitan monoester. In some embodiments, the acyl chains of sorbitan diester or sorbitan triester will be saturated.

[0130] Usually, sorbitan esters (such as sorbitan monoester) have a hydrophilic-lipophilic balance (HLB) value of 1 to 9. In some embodiments, sorbitan esters (such as sorbitan monoester) have an HLB value of 1 to 5. In some embodiments, the hydrophobic surfactant has an HLB value of about 4 to 5.

[0131] The exemplary sorbitan diester used in this specification can be represented as a compound of formula IV below or its stereoisomer (for example, in the formula, R is saturated or unsaturated C1-C30 Alkyl alkyl groups, preferably saturated or unsaturated C1-C 20 Alkyl alkyl groups, more preferably saturated or unsaturated C 10 ~C 20 It is an alkyl group, R 1 At least one of them is H, but the others are -C(=O)Y, where Y is saturated or unsaturated C1~C 30 Alkyl alkyl groups, preferably saturated or unsaturated C1-C 20 Alkyl alkyl groups, more preferably saturated or unsaturated C 10 ~C 20 (It is an alkyl group). In exemplary embodiments, the alkyl group is an acyclic alkyl group. Exemplary sorbitane diesters also include positional isomers of formula IV. As will be understood by those skilled in the art, exemplary sorbitane diesters may be formula IV and salt forms of its stereoisomers or positional isomers (e.g., pharmaceutically acceptable salts). [ka] Formula IV

[0132] Exemplary sorbitan triesters used herein may be represented as compounds of the following formula V or their stereoisomers (e.g., formulas Va, Vb, or Vc, but not limited to these), where R is saturated or unsaturated C1-C13 30 Alkyl alkyl groups, preferably saturated or unsaturated C1-C 20 Alkyl alkyl groups, more preferably saturated or unsaturated C 10 ~C 20 It is an alkyl group, R 1 is -C(=O)Y, where Y may be the same or different in each case, and C1~C may be saturated or unsaturated. 30 Alkyl alkyl groups, preferably saturated or unsaturated C1-C 20 Alkyl alkyl groups, more preferably saturated or unsaturated C 10 ~C 20It is an alkyl group. In exemplary embodiments, the alkyl group is an acyclic alkyl group. Exemplary sorbitan triesters also include positional isomers of formulas V, Va, Vb, or Vc (for example, the hydroxyl functional group is an ester functional group (for example, the alkyl is saturated or unsaturated C1-C) 30 Alkyl alkyl groups, preferably saturated or unsaturated C1-C 20 Alkyl alkyl groups, more preferably saturated or unsaturated C 10 ~C 20 It is substituted with an alkyl group (an alkyl ester), and one of the alkyl esters (e.g., a cyclic alkyl ester or an acyclic alkyl ester) is substituted with a hydroxyl functional group. As will be understood by those skilled in the art, exemplary sorbitan triesters may be formulas V, Va, Vb, or Vc, and salts of their stereoisomers or positional isomers (e.g., pharmaceutically acceptable salts). [ka]

[0133] With regard to stereoisomers, it will be understood by those skilled in the art that the sorbitan ester has a chiral center and can exist, for example, as a racemic compound, as a racemic mixture, and as individual enantiomers and diastereomers.

[0134] In embodiments where the sorbitan-based nonionic surfactant is a sorbitan ester, the NLC-based composition or formulation is typically, for example, about 0.1% to about 15% sorbitan ester (w / v), 0.1% to about 10% sorbitan ester (w / v), 0.1% to about 5% sorbitan ester (w / v), about 0.1% to about 4% sorbitan ester (w / v), about 0.1% to about 4% sorbitan ester (w / v), about 0.1% to about 2.5% sorbitan ester (w / v), about 0.1% to about 2% sorbitan ester (w / v), 0.1% to about 1.5% sorbitan ester ( It typically contains sorbitan ester (w / v) in amounts of 0.1% to about 1%, sorbitan ester (w / v) in amounts of 0.1% to about 0.5%, sorbitan ester (w / v) in amounts of 0.3% to about 2.5%, sorbitan ester (w / v) in amounts of 0.3% to about 2%, sorbitan ester (w / v) in amounts of 0.3% to about 1.5%, sorbitan ester (w / v) in amounts of 0.3% to about 1.5%, or any other content or range of sorbitan ester as described herein, for example, about 0.25% to about 15%. In some embodiments, the NLC-based composition contains about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, or about 4% (w / v) of sorbitan ester. Higher or lower w / v% are intended herein, especially when considering diluted or concentrated formulations.

[0135] That is, if the sorbitan ester is a sorbitan monoester (e.g., SPAN60®, SPAN80®), then the NLC-based composition or formulation may contain, for example, about 0.1% to about 15% sorbitan monoester (w / v), 0.1% to about 10% sorbitan monoester (w / v), 0.1% to about 5% sorbitan monoester (w / v), about 0.1% to about 4% sorbitan monoester (w / v), about 0.1% to about 4% sorbitan monoester (w / v), about 0.1% to about 2.5% sorbitan monoester (w / v), about 0.1% to about 2% sorbitan monoester (w / v), or 0.1% to about 1.5% sorbitan monoester (w / v). It typically contains sorbitan monoester (w / v), 0.1% to about 1% sorbitan monoester (w / v), 0.1% to about 0.5% sorbitan monoester (w / v), 0.3% to about 2.5% sorbitan monoester (w / v), about 0.3% to about 2% sorbitan monoester (w / v), 0.3% to about 1.5% sorbitan monoester (w / v), 0.3% to about 1% sorbitan monoester (w / v), or 0.3% to about 0.5% sorbitan monoester (w / v), or any other content or range of sorbitan monoester as described herein, for example, about 0.25% to about 15% sorbitan monoester. In some embodiments, the NLC-based composition or formulation contains about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, or about 4% (w / v) of sorbitan monoester. Higher or lower w / v% are intended herein, especially when considering diluted or concentrated formulations.

[0136] That is, if the sorbitan ester is a sorbitane diester, the NLC-based composition or formulation may contain, for example, about 0.1% to about 15% sorbitane diester (w / v), 0.1% to about 10% sorbitane diester (w / v), 0.1% to about 5% sorbitane diester (w / v), about 0.1% to about 4% sorbitane diester (w / v), about 0.1% to about 4% sorbitane diester (w / v), about 0.1% to about 2.5% sorbitane diester (w / v), about 0.1% to about 2% sorbitane diester (w / v), or 0.1% to about 1.5% sorbitane diester (w / v). It typically contains 0.1% to about 1% sorbitane diester (w / v), 0.1% to about 0.5% sorbitane diester (w / v), 0.3% to about 2.5% sorbitane diester (w / v), about 0.3% to about 2% sorbitane diester (w / v), 0.3% to about 1.5% sorbitane diester (w / v), 0.3% to about 1% sorbitane diester (w / v), or 0.3% to about 0.5% sorbitane diester (w / v), or any other content or range of sorbitane diester as described herein, for example, about 0.25% to about 15% sorbitane diester. In some embodiments, the NLC-based composition or formulation contains about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, or about 4% (w / v) of sorbitane diester. Higher or lower w / v% are intended herein, especially when considering diluted or concentrated formulations.

[0137] Therefore, if the sorbitan ester is a sorbitan triester (e.g., SPAN85® or SPAN65®), the NLC-based composition or formulation may contain, for example, about 0.1% to about 15% sorbitan triester (w / v), 0.1% to about 10% sorbitan triester (w / v), 0.1% to about 5% sorbitan triester (w / v), about 0.1% to about 4% sorbitan triester (w / v), about 0.1% to about 4% sorbitan triester (w / v), about 0.1% to about 2.5% sorbitan triester (w / v), about 0.1% to about 2% sorbitan triester (w / v), or 0.1% to about 1.5% sorbitan triester (w / v). It typically contains sorbitan triester (w / v), 0.1% to about 1% sorbitan triester (w / v), 0.1% to about 0.5% sorbitan triester (w / v), 0.3% to about 2.5% sorbitan triester (w / v), about 0.3% to about 2% sorbitan triester (w / v), 0.3% to about 1.5% sorbitan triester (w / v), 0.3% to about 1% sorbitan triester (w / v), or 0.3% to about 0.5% sorbitan triester (w / v), or any other content or range of sorbitan triester as described herein, for example, about 0.25% to about 15%. In some embodiments, the NLC-based composition or formulation contains about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, or about 4% (w / v) of sorbitan triester. Higher or lower w / v% are intended herein, especially when considering diluted or concentrated formulations.

[0138] In exemplary embodiments, the sorbitan ester (e.g., sorbitan monoester, sorbitan diester, or sorbitan triester) is present in an amount sufficient to enhance the composition's ability to promote the delivery and / or expression of the bioactive agent (e.g., RNA) compared to a control composition that does not contain the sorbitan ester (e.g., sorbitan monoester, sorbitan diester, or sorbitan triester, respectively). In embodiments in which the composition is administered to the subject in an effective amount, the composition may induce an antibody titer against the antigen that is higher than the antibody titer induced when the subject is administered a control composition that does not contain the sorbitan ester, or when the bioactive agent is administered to the subject without this NLC. In some embodiments, the composition induces an immune response (e.g., neutralizing antibody titer) in the subject that is higher than the immune response induced in the subject by the control composition that does not contain the sorbitan ester. The immune response may be, for example, an innate immune response, a cellular immune response, or an antibody response. The neutralizing antibody titer may be measured by any assay known to those skilled in the art, including, but not limited to, plaque reduction neutralizing titration (Ratnam, S et al. J. Clin. Microbiol (2011), 33 (4): 811-815; Timiryazova, T et al. Am J Trop Med Hyg (2013), 88(5): 962-970).

[0139] D. Surfactants

[0140] The NLC described herein includes surfactants in addition to the sorbitan-based nonionic surfactant (e.g., sorbitan ester). Several surfactants specifically designed for use in biological applications exist. Such surfactants are classified into four basic types, which can be used in the present invention (anionic surfactants, cationic surfactants, zwitterionic surfactants, and nonionic surfactants). A particularly useful group of surfactants are hydrophilic nonionic surfactants, in particular polyoxyethylene sorbitan monoesters and polyoxyethylene sorbitan triesters. These substances are called polysorbates and are commercially available under the trade name TWEEN®, and are useful in the preparation of this NLC. TWEEN® surfactants typically have HLB values ​​in the range of 9.6 to 16.7. TWEEN® surfactants are commercially available. Other nonionic surfactants that can be used include, for example, polyoxyethylene fatty acid ethers derived from lauryl alcohol, polyoxyethylene fatty acid ethers derived from acetyl alcohol, polyoxyethylene fatty acid ethers derived from stearyl alcohol, and polyoxyethylene fatty acid ethers derived from oleyl alcohol, polyoxyethylene fatty acids produced by the reaction of ethylene oxide with long-chain fatty acids, polyoxyethylene, polyol fatty acid esters, polyoxyethylene ethers, polyoxypropylene fatty acid ethers, polyoxyethylene-containing beeswax derivatives, polyoxyethylene lanolin derivatives, polyoxyethylene fatty acid glycerides, polyoxyethylene glycerol fatty acid esters, or C 12~22 These are other polyoxyethylene fatty acid derivatives, alcohol derivatives, or ether derivatives of long-chain fatty acids.

[0141] In some embodiments, it is preferable to select a nonionic surfactant having an HLB value in the range of about 7 to 16. This value may be obtained by using a single nonionic surfactant, such as a TWEEN® surfactant, or it may be achieved by using a mixture of surfactants. In some embodiments, the NLC comprises a single nonionic surfactant as a nonionic surfactant to stabilize the emulsion, most often particularly a TWEEN® surfactant. In an exemplary embodiment, the emulsion contains TWEEN® 80 (also known as polysorbate 80).

[0142] The NLC-based composition or formulation may contain, for example, about 0.01% to about 15% surfactant (w / v), about 0.01% to about 10% surfactant (w / v), about 0.01% to about 5% surfactant (w / v), about 0.01% to about 2.5% surfactant, about 0.01% to about 2% surfactant, 0.01% to about 1.5% surfactant, 0.01% to about 1% surfactant, 0.01% to about 0.5% surfactant, or 0.05% to about 0.5% The formulation may contain surfactants, surfactants in concentrations of 0.08% to about 0.5%, about 0.08%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, or any other concentrations or ranges of surfactants as described herein. Higher or lower w / v% are intended herein, especially when considering diluted or concentrated formulations.

[0143] The NLC of the present invention may contain additional components, such as components that promote NLC formation, components that improve complex formation between negatively charged molecules and cationic particles, components that promote the proper release of negatively charged molecules (such as RNA molecules), and / or components that increase the stability of negatively charged molecules (e.g., prevent the degradation of RNA molecules).

[0144] The aqueous phase (continuous phase) of this NLC is usually a buffer salt solution (e.g., physiological saline) or water. The buffer salt solution is usually an aqueous solution containing salt (e.g., NaCl) and buffer (e.g., citrate buffer), and may further contain, for example, osmolality adjusters (e.g., sugars), polymers, surfactants, or combinations thereof. When formulating an emulsion for parenteral administration, it is preferable to configure the final buffer so that the tonicity, i.e., osmolality, is essentially the same as that of a normal physiological solution, in order to prevent undesirable post-administration events such as swelling or rapid absorption of the composition after administration. It is also preferable to buffer the aqueous phase to maintain a pH that is compatible with normal physiological conditions. In some cases, it may be preferable to maintain the pH at a specific level to ensure the stability of certain components of this NLC. For example, it may be preferable to prepare an NLC that is isotonic (i.e., the same permeable solute (e.g., salt) concentration as normal cells in the body and blood) and isosmotic. To adjust the tonicity, this NLC may contain physiological salts (such as sodium salts). In some embodiments, for example, about 0.9% (w / v) sodium chloride (NaCl) (physiological saline) may be used. Other salts that may be included include, for example, potassium chloride, potassium dihydrogen phosphate, disodium phosphate, magnesium chloride, and calcium chloride. The tonicity can also be adjusted using a nonionic tonicifying agent. In this invention, monosaccharides classified as aldoses, such as glucose, mannose, arabinose, and ribose, and monosaccharides classified as ketoses, such as fructose, sorbose, and xylulose, can be used as nonionic tonicifying agents. Disaccharides such as sucrose, maltose, trehalose, and lactose can also be used. Furthermore, alditols (acyclic polyhydric alcohols, also known as sugar alcohols), such as glycerol, mannitol, xylitol, and sorbitol, are also nonionic tonicifying agents that may be useful in this invention. The nonionic tonicity regulator can be present at a concentration of, for example, about 0.1% to about 10% or about 1% to about 10%, depending on the nonionic tonicity regulator used.

[0145] The aqueous phase may be treated with a buffer. Any buffer solution that is physiologically acceptable may be used herein, such as water, citrate buffer, phosphate buffer, acetate buffer solution, Tris buffer, bicarbonate buffer, carbonate buffer, succinate buffer, and the like. Preferably, the pH of the aqueous component is 4.0 to 8.0 or about 4.5 to about 6.8. In another exemplary embodiment, the aqueous phase, or the buffer solution, is prepared using water free of RNase or water treated with DEPC. In some examples, buffers with high salt concentrations are avoided because high salt concentrations in the buffer may prevent the formation of complexes between negatively charged molecules and emulsion particles. In other examples, the buffer solution may contain a specific amount of salt.

[0146] In an exemplary embodiment, the buffer is a 10 mM citrate buffer (e.g., sodium citrate) having a pH of about 5.0 to 8.0. In another exemplary embodiment, the aqueous phase, or the buffer solution, is prepared using water free of RNase or water treated with DEPC. In other exemplary embodiments, the composition of the present invention does not contain a citrate buffer.

[0147] The aqueous phase may contain additional components such as a molecule that changes the osmolality of the aqueous phase or a molecule that stabilizes the negatively charged molecule after complex formation. The osmolality of the aqueous phase is preferably adjusted using a nonionic tonicity enhancer such as a sugar (e.g., trehalose, sucrose, dextrose, fructose, reduced palatinose, etc.), a sugar alcohol (e.g., mannitol, sorbitol, xylitol, erythritol, lactitol, maltitol, glycerol, etc.), or a combination thereof. If necessary, a nonionic polymer (e.g., poly(alkyl glycol) such as polyethylene glycol, polypropylene glycol, or polybutylene glycol) or a nonionic surfactant can be used.

[0148] E. Oil / surfactant ratio

[0149] An exemplary NLC comprises a hydrophobic core containing liquid oil and solid lipids, and a surfactant (also known as an emulsifier or emulsion agent) that constitutes an interface separating the hydrophobic phase (liquid oil and solid lipids, collectively referred to as oil herein) from the aqueous phase. The surfactant is typically present on the surface of the NLC nanoparticles, and its content determines the total available surface area. The oil, on the other hand, is present in the core and primarily contributes to the total available volume. As a result, an increase in the surfactant / oil ratio increases the surface area (SA) / volume (V) ratio, i.e., if the volume of the substance is fixed, an increase in the SA / V ratio means a decrease in the NLC particle size. Exemplary NLC compositions can also be described in terms of the molar ratios of the various components, instead of, or in addition to, describing the weight volume percentages of the various components. In some embodiments, the exemplary NLCs of the present invention have oil:surfactant molar ratios of about 0.05 to about 12, about 0.05 to about 9, about 0.05 to about 8, about 0.05 to about 1, or about 0.1 to about 1. The inventors have shown that smaller NLCs can be synthesized by reducing the oil:surfactant molar ratio. Furthermore, by reducing the oil content in the NLC, the potential toxicity of the formulation can be reduced. In other embodiments, exemplary NLCs of the present invention have oil:surfactant molar ratios of about 0.5 to about 12, about 0.5 to about 9, 1 to about 9, about 2 to about 9, about 3 to about 9, about 4 to about 9, about 4.5 to about 9, or about 4.5 or about 5 to about 7. Exemplary formulations have oil:surfactant molar ratios of about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12. When used herein, the oil:surfactant molar ratio is determined by (i) adding the number of moles of lipids constituting the oily core (solid phase lipids and liquid phase lipids) to ensure the number of moles of lipids in the oily core, (ii) adding the number of moles of cationic components (e.g., DOTAP), hydrophobic surfactants (e.g., sorbitan esters), and hydrophilic surfactants (Tween 80) to ensure the number of moles of surfactant, and (iii) dividing the number of moles of lipids in the oily core by the number of moles of surfactant.

[0150] F. Hydrophilic surfactant: Cationic component ratio

[0151] The inventors have found that the ratio of hydrophilic surfactant to cationic component affects the protective effect of the NLC against RNAase degradation and can affect the immunogenicity of the formulation. In particular, the inventors have found that a Tween:DOTAP ratio of about 0.6 is optimal for obtaining consistent results in the delivery and expression of RNA bioactivators, while a Tween:DOTAP ratio of about 2.0 or higher is not optimal for obtaining such consistency. Accordingly, exemplary NLCs of the present invention have a hydrophilic surfactant / cateionic component (e.g., cationic lipid) ratio of about 0.2 to about 1.5, about 0.2 to about 1, or about 0.5 to about 1. When Tween and DOTAP are present in the composition, exemplary NLCs of the present invention have a Tween:DOTAP ratio of about 0.2 to about 1.5, about 0.2 to about 1, or about 0.5 to about 1. When used herein, the hydrophilic surfactant:cationic component ratio is determined by (i) adding the number of moles of hydrophilic surfactant to ensure the value of the number of moles of hydrophilic surfactant, (ii) adding the number of moles of cationic component to ensure the value of the number of moles of cationic component, and (iii) dividing the number of moles of hydrophilic surfactant by the number of moles of cationic component.

[0152] G. Load capacity

[0153] The inventors have found that the amount of RNA (NLC) packed into an NLC formulation can be manipulated by adjusting the ratio of hydrophilic surfactant to cationic component and the amount of oil present in the formulation, thereby reducing the average NLC particle size. Typical RNA packed into NLC formulations are at least about 10 μg / mL of RNA, at least about 20 μg / mL, at least about 50 μg / mL, at least about 100 μg / mL, at least about 200 μg / mL, at least about 300 μg / mL, or at least about 400 μg / mL. Typically, NLC formulations with average particle sizes of 20 nm to about 110 nm, about 20 nm to about 80 nm, about 20 nm to about 70 nm, and about 20 nm to about 60 nm show an increase in packed amount.

[0154] H. Exemplary nanostructured supports and their weight volume percentage

[0155] In selected exemplary embodiments, the NLC composition comprises about 0.2 w / v% to about 40 w / v% of liquid-phase lipids, about 0.02 w / v% to about 10 w / v% of solid-phase lipids, about 0.2 w / v% to about 10 w / v% of cationic lipids, about 0.25 w / v% to about 5 w / v% of hydrophobic surfactants (e.g., sorbitan esters), and about 0.2 w / v% to about 10 w / v%, about 0.2 w / v% to about 5 w / v%, about 0.5 w / v% to about 5 w / v%, or about 0.5 w / v% to about 10 w / v% of hydrophilic surfactants. This NLC composition is referred to herein as Formulation A. In all embodiments of formulation A, the hydrophilic surfactant may be present in amounts of 0.2 w / v% to about 10 w / v%, 0.2 w / v% to about 5 w / v%, 0.5 w / v% to about 5 w / v%, or about 0.5 w / v% to about 10 w / v%.

[0156] In selected exemplary embodiments, the NLC composition comprises about 0.2 w / v% to about 40 w / v% of liquid-phase lipids, about 0.1 w / v% to about 10 w / v% of solid-phase lipids, about 0.2 w / v% to about 10 w / v% of cationic lipids, about 0.25 w / v% to about 5 w / v% of hydrophobic surfactants (e.g., sorbitan esters), and about 0.2 w / v% to about 10 w / v%, about 0.2 w / v% to about 5 w / v%, about 0.5 w / v% to about 5 w / v%, or about 0.5 w / v% to about 10 w / v% of hydrophilic surfactants. This NLC composition is referred to herein as Formulation B. In all embodiments of formulation B, the hydrophilic surfactant may be present in amounts of 0.2 w / v% to about 10 w / v%, 0.2 w / v% to about 5 w / v%, 0.5 w / v% to about 5 w / v%, or about 0.5 w / v% to about 10 w / v%.

[0157] In selected exemplary embodiments, the NLC composition comprises about 0.2 w / v% to about 1 w / v% of liquid-phase lipids, about 0.02 w / v% to about 1 w / v% of solid-phase lipids, about 2 w / v% to about 10 w / v% of cationic lipids, about 2 w / v% to about 5 w / v% of sorbitan esters, and about 2 w / v% to about 5 w / v% of hydrophilic surfactants. This NLC composition is referred to herein as Formulation C.

[0158] In selected exemplary embodiments, the NLC composition comprises about 2 w / v% to about 40 w / v% liquid-phase lipids, about 0.1 w / v% to about 10 w / v% solid-phase lipids, about 0.2 w / v% to about 10 w / v% cationic lipids, about 0.25 w / v% to about 5 w / v% hydrophobic surfactants (e.g., sorbitan esters), and about 0.2 w / v% to about 10 w / v%, about 0.2 w / v% to about 5 w / v%, about 0.5 w / v% to about 5 w / v%, or about 0.5 w / v% to about 10 w / v% hydrophilic surfactants. This NLC composition is referred to herein as Formulation D. In all embodiments of formulation D, the hydrophilic surfactant may exist as a hydrophilic surfactant in amounts of 0.2 w / v% to about 10 w / v%, 0.2 w / v% to about 5 w / v%, about 0.5 w / v% to about 5 w / v%, or about 0.5 w / v% to about 10 w / v%.

[0159] In selected exemplary embodiments, the NLC composition comprises about 2 w / v% to about 10 w / v% of liquid-phase lipids, about 0.1 w / v% to about 10 w / v% of solid-phase lipids, about 0.2 w / v% to about 10 w / v% of cationic lipids, about 0.25 w / v% to about 5 w / v% of sorbitan esters, and about 0.2 w / v% to about 10 w / v% or about 0.2 w / v% to about 5 w / v% of a hydrophilic surfactant. This NLC composition is referred to herein as Formulation E. In any embodiment of Formulation E, the hydrophilic surfactant may be present in an amount of 0.2 w / v% to about 10 w / v% or about 0.2 w / v% to about 5 w / v%.

[0160] In selected exemplary embodiments, the NLC composition comprises about 2 w / v% to about 10 w / v% liquid-phase lipids, about 0.1 w / v% to about 3 w / v% solid-phase lipids, about 1 w / v% to about 5 w / v% cationic lipids, about 1 w / v% to about 5 w / v% sorbitan esters, and about 1 w / v% to about 5 w / v% hydrophilic surfactants. This NLC composition is referred to herein as Formulation F.

[0161] In selected exemplary embodiments, the NLC composition comprises about 2 w / v% to about 5 w / v% liquid-phase lipids, about 0.1 w / v% to about 2 w / v% solid-phase lipids, about 2 w / v% to about 5 w / v% cationic lipids, about 2 w / v% to about 5 w / v% sorbitan esters, and about 2 w / v% to about 5 w / v% hydrophilic surfactants. This NLC composition is referred to herein as Formulation G.

[0162] In selected exemplary embodiments, the NLC composition comprises about 2 w / v% to about 10 w / v% of liquid-phase lipids, about 0.1 w / v% to about 3 w / v% of solid-phase lipids, about 0.2 w / v% to about 2 w / v% of cationic lipids, about 0.25 w / v% to about 2 w / v% of sorbitan esters, and about 0.2 w / v% to about 5 w / v% or about 0.5 w / v% to about 5 w / v% of a hydrophilic surfactant. This NLC composition is referred to herein as Formulation H. In any embodiment of Formulation H, the hydrophilic surfactant may be present in an amount of about 0.2% to about 5 w / v% or about 0.5 w / v% to about 5 w / v%.

[0163] In selected exemplary embodiments, the NLC composition comprises about 2 w / v% to about 6 w / v% of liquid-phase lipids, about 0.1 w / v% to about 1 w / v% of solid-phase lipids, about 0.2 w / v% to about 1 w / v% of cationic lipids, about 0.25 w / v% to about 1 w / v% of sorbitan esters, and about 0.2 w / v% to about 5 w / v% or about 0.5 w / v% to about 5 w / v% of a hydrophilic surfactant. This NLC composition is referred to herein as Formulation I. In any embodiment of Formulation I, the hydrophilic surfactant may be present in an amount of about 0.2% to about 5 w / v% or about 0.5 w / v% to about 5 w / v%.

[0164] In selected exemplary embodiments, the NLC composition comprises about 2 w / v% to about 6 w / v% of liquid-phase lipids, about 0.1 w / v% to about 1 w / v% of solid-phase lipids, about 0.2 w / v% to about 1 w / v% of cationic lipids, about 0.25 w / v% to about 1 w / v% of sorbitan esters, and about 0.2 w / v% to about 2 w / v% or about 0.2 w / v% to about 1 w / v% of hydrophilic surfactants. This NLC composition is referred to herein as Formulation J.

[0165] In selected exemplary embodiments, the NLC composition comprises about 2 w / v% to about 6 w / v% of liquid-phase lipids, about 0.1 w / v% to about 1 w / v% of solid-phase lipids, about 0.2 w / v% to about 1 w / v% of cationic lipids, about 0.25 w / v% to about 1 w / v% of sorbitan esters, and about 0.2 w / v% to about 0.5 w / v% or 0.2 w / v% to about 1 w / v% of a hydrophilic surfactant. This NLC composition is referred to herein as Formulation K. In any embodiment of Formulation K, the hydrophilic surfactant may be present in an amount of about 0.2 w / v% to about 0.5 w / v% or 0.2 w / v% to about 1 w / v%.

[0166] In selected exemplary embodiments, the NLC composition comprises about 10 w / v% to about 40 w / v% liquid-phase lipids, about 1% to about 2% solid-phase lipids, about 2% to about 5% cationic lipids, about 3 w / v% to about 5 w / v% sorbitan esters, and about 3 w / v% to about 5 w / v% hydrophilic surfactants. This NLC composition is referred to herein as Formulation L.

[0167] In selected exemplary embodiments, the NLC composition comprises about 10 w / v% to about 20 w / v% liquid-phase lipids, about 0.5% to about 1.5% solid-phase lipids, about 3% to about 4% cationic lipids, about 3 w / v% to about 4 w / v% sorbitan esters, and about 3 w / v% to about 5 w / v% hydrophilic surfactants. This NLC composition is referred to herein as Formulation M.

[0168] In selected exemplary embodiments, the NLC composition comprises about 15 w / v% liquid-phase lipids, about 1% solid-phase lipids, about 3% cationic lipids, about 3.7 w / v% sorbitan esters, and about 3.7 w / v% hydrophilic surfactants. This NLC composition is referred to herein as Formulation N.

[0169] In selected exemplary embodiments, the NLC composition comprises about 30 w / v% liquid-phase lipids, about 1.8% solid-phase lipids, about 3% cationic lipids, about 3.7 w / v% sorbitan ester, and about 3.7 w / v% hydrophilic surfactant. This NLC composition is referred to herein as Formulation O.

[0170] In selected exemplary embodiments, the NLC composition comprises about 3 w / v% to about 4 w / v% liquid-phase lipids, about 0.2% to about 1% solid-phase lipids, about 3% to about 5% cationic lipids, about 3% to about 5% sorbitan esters, and about 3% to about 5% hydrophilic surfactants. This NLC composition is referred to herein as Formulation P.

[0171] In selected exemplary embodiments, the NLC composition comprises about 0.2 w / v% to about 40 w / v% liquid-phase lipids, about 0.1 w / v% to about 10 w / v% solid-phase lipids, about 0.2 w / v% to about 10 w / v% cationic lipids, about 0.25 w / v% to about 15 w / v% sorbitan monoester, and about 0.5 w / v% to about 15 w / v% hydrophilic surfactant. This NLC composition is referred to herein as Formulation Q.

[0172] In selected exemplary embodiments, the NLC composition comprises about 4 w / v% liquid-phase lipids, about 0.25 w / v% solid-phase lipids, about 0.4 w / v% cationic lipids, about 0.5 w / v% sorbitan ester, and about 2 w / v% hydrophilic surfactant. This NLC composition is referred to herein as Formulation R.

[0173] In selected exemplary embodiments, the NLC composition comprises about 4 w / v% liquid-phase lipids, about 0.25 w / v% solid-phase lipids, about 0.4 w / v% cationic lipids, about 0.5 w / v% sorbitan ester, and about 0.5 w / v% hydrophilic surfactant. This NLC composition is referred to herein as Formulation S.

[0174] In selected exemplary embodiments, the NLC composition comprises about 3.75 w / v% liquid-phase lipids, about 0.25 w / v% solid-phase lipids, about 3 w / v% cationic lipids, about 3.7 w / v% sorbitan ester, and about 3.7 w / v% hydrophilic surfactant. This NLC composition is referred to herein as Formulation T.

[0175] In selected exemplary embodiments, the NLC composition comprises about 3.75 w / v% liquid-phase lipids, about 0.25 w / v% solid-phase lipids, about 1.5 w / v% cationic lipids, about 3.7 w / v% sorbitan ester, and about 1.5 w / v% hydrophilic surfactant. This NLC composition is referred to herein as Formulation U.

[0176] In selected exemplary embodiments, the NLC composition comprises about 4.75 w / v% liquid-phase lipids, about 0.25 w / v% solid-phase lipids, about 0.5 w / v% cationic lipids, about 0.5 w / v% sorbitan ester, and about 0.4 w / v% hydrophilic surfactant. This NLC composition is referred to herein as Formulation V.

[0177] In selected exemplary embodiments, the NLC composition comprises about 0.2 w / v% to about 40 w / v% of liquid-phase lipids, about 0.2 w / v% to about 10 w / v% of cationic lipids, about 0.25 w / v% to about 5 w / v% of hydrophobic surfactants (e.g., sorbitan esters), and about 0.2 w / v% to about 10 w / v%, about 0.2 w / v% to about 5 w / v%, about 0.5 w / v% to about 5 w / v%, or about 0.5 w / v% to about 10 w / v% of hydrophilic surfactants. This NLC composition is referred to herein as Formulation W. In all embodiments of formulation W, the hydrophilic surfactant may be present in amounts of 0.2 w / v% to about 10 w / v%, 0.2 w / v% to about 5 w / v%, 0.5 w / v% to about 5 w / v%, or about 0.5 w / v% to about 10 w / v%.

[0178] Those skilled in the art will understand that any of the NLC compositions / formulations described herein, including formulations A to W, can be diluted or concentrated when used in the present invention. For example, when mixed with a bioactive agent for delivery, the formulation may be diluted during the mixing process. The NLC composition / formulation may be diluted, for example, 1:2. All NLC compositions / formulations described herein may be diluted, for example, about 2 to about 500 times, preferably about 2 to about 100 times. Dilution is usually, but not always, performed when the formulation is mixed with a bioactive agent (e.g., RNA or DNA) for delivery. The NLC composition / formulation may be diluted, for example, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 100 times, or about 500 times. As will be understood by those skilled in the art, diluted formulations of the present invention have reduced concentrations of liquid-phase lipids, solid-phase lipids, cationic components, hydrophobic surfactants (e.g., sorbitan esters), and surfactants (e.g., hydrophilic surfactants), but the ratios of liquid-phase lipids to solid-phase lipids, cationic components to hydrophobic surfactants (e.g., sorbitan esters), and surfactants remain the same. The present invention provides not only formulations A to W, but also diluted forms of formulations A to W. In some cases, the diluted formulations bind with bioactive agents (e.g., form complexes). For example, particularly preferred formulations are two-fold diluted formulation S or formulation T. Such diluted formulation S contains about 2 w / v% liquid-phase lipids, about 0.13 w / v% solid-phase lipids, about 0.2 w / v% cationic lipids, about 0.25 w / v% sorbitan esters, and about 0.25 w / v% hydrophilic surfactant. Such a diluted formulation T comprises approximately 1.88 w / v% liquid-phase lipids, approximately 0.13 w / v% solid-phase lipids, approximately 1.5 w / v% cationic lipids, approximately 1.85 w / v% sorbitan ester, and approximately 1.85 w / v% hydrophilic surfactant.

[0179] Alternatively, the composition / formulation may be concentrated, for example, about 2 to about 30 times, preferably about 2 to about 20 times. The composition / formulation may be concentrated, for example, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 15 times (old), about 20 times, about 25 times, or about 30 times. That is, the present invention provides not only formulations A to W, but also concentrated forms of formulations A to U.

[0180] With respect to any of the NLC formulations of the present invention comprising formulations A to V, including diluted and concentrated formulations of formulations A to V, the following and any combination thereof may apply: (i) the sorbitan ester is a sorbitan monoester, sorbitan diester, or sorbitan triester; (ii) the sorbitan ester is a sorbitan monoester selected from sorbitan monostearate, sorbitan monooleate, or sorbitan monolaurate, or sorbitan trioleate or sorbitan triester (iii) The liquid phase lipid is a sorbitan triester selected from aretes; (iv) The solid phase lipid is a glycerolipid; (v) The solid phase lipid is a microcrystalline triglyceride; (vi) The solid phase lipid is trimiristin; (vii) The cationic lipid is DOTAP; (viii) The hydrophilic surfactant is polysorbate 80 (also known as Tween 80); (ix) The sorbitan ester is a monoester, the liquid phase lipid is squalene, and the solid phase lipid is a glycerolipid; (x) The so (xi) The sorbitan ester is a monoester, the liquid phase lipid is squalene; the solid phase lipid is a glycerolipid; the cationic lipid is DOTAP, and the hydrophilic surfactant is polysorbate 80; (xi) The sorbitan ester is sorbitan monostearate or sorbitan monooleate or sorbitan monolaurate, the liquid phase lipid is squalene; the solid phase lipid is trimyristine; the cationic lipid is DOTAP, and the hydrophilic surfactant is polysorbate 80; (xii) (xiii) The sorbitan ester is a triester, the liquid-phase lipid is squalene, and the solid-phase lipid is a glycerolipid; (xiv) The sorbitan ester is a triester, the liquid-phase lipid is squalene, and the solid-phase lipid is a glycerolipid; the cationic lipid is DOTAP, and the hydrophilic surfactant is polysorbate 80; (xiv) The sorbitan ester is sorbitan trioleate or sorbitan tristearate, the liquid-phase lipid is squalene, and the solid-phase lipid is trimiristin;The cationic lipid is DOTAP, and the hydrophilic surfactant is polysorbate 80.

[0181] The present invention also provides formulations A to Q, including those shown in paragraph

[0180] above, wherein the oil / surfactant ratio is about 0.05 to about 12, or about 0.05 to about 9, or about 0.05 to about 8, or about 0.05 to about 1, or about 0.1 to about 1. The present invention also provides formulations A to Q, including those shown in paragraph

[0180] above, wherein the oil:surfactant molar ratio is about 0.5 to about 12, about 1 to about 9, about 2 to about 9, about 3 to about 9, about 4 to about 9, about 4.5 to about 9, or about 4.5 to about 5 to about 7.

[0182] The present invention provides formulations A to Q, which include the above, wherein the hydrophilic surfactant / cationic component (e.g., cationic lipid) ratio is about 0.2 to about 1, or about 0.5 to about 1.

[0183] That is, some exemplary NLC compositions are formulations A, B, or Q, diluted or concentrated, with an oil:surfactant molar ratio of about 0.5 to about 12, about 1 to about 9, about 2 to about 9, about 3 to about 9, about 4 to about 9, about 4.5 to about 9, or about 4.5 or about 5 to about 7, and a hydrophilic surfactant / cationic component (e.g., cationic lipid) ratio of about 0.2 to about 1.5.

[0184] That is, some exemplary NLC compositions are formulations A, B, or Q, diluted or concentrated, with an oil:surfactant molar ratio of about 0.5 to about 12, about 1 to about 9, about 2 to about 9, about 3 to about 9, about 4 to about 9, about 4.5 to about 9, or about 4.5 or about 5 to about 7, and a hydrophilic surfactant / cationic component (e.g., cationic lipid) ratio of about 0.2 to about 1.

[0185] Some exemplary NLC compositions are formulations A, B, or Q, diluted or concentrated, with oil:surfactant molar ratios of about 0.5 to about 12, about 1 to about 9, about 2 to about 9, about 3 to about 9, about 4 to about 9, about 4.5 to about 9, or about 4.5 or about 5 to about 7, and a hydrophilic surfactant / cationic component (e.g., cationic lipid) ratio of about 0.5 to about 1.

[0186] Some exemplary NLC compositions are formulations A, B, or Q, diluted or concentrated, having an oil:surfactant molar ratio of 0.05 to about 12 or about 0.05 to about 9 or about 0.05 to about 8 or about 0.05 to about 1 or about 0.1 to about 1, and a hydrophilic surfactant / cationic component (e.g., cationic lipid) ratio of about 0.5 to about 1.5.

[0187] Some exemplary NLC compositions are formulations A, B, or Q, diluted or concentrated, having an oil:surfactant molar ratio of 0.05 to about 12 or about 0.05 to about 9 or about 0.05 to about 8 or about 0.05 to about 1 or about 0.1 to about 1, and a hydrophilic surfactant / cationic component (e.g., cationic lipid) ratio of about 0.5 to about 1.

[0188] The present invention provides formulations A to W, which include all formulations described in paragraphs

[0180] to

[0187] , wherein the average particle size of the NLC particles is about 40 nm or 50 nm to about 80 nm, about 40 nm or 50 nm to about 70 nm, and about 40 nm or 50 nm to about 60 nm.

[0189] III. Physiological characteristics of nanostructured lipid carriers

[0190] A. Size

[0191] The size of the NLC can be evaluated by methods known in the art, including, but not limited to, X-ray diffraction, laser diffraction, dynamic light scattering (DLS), cryo-electron microscopy (CryoEM), or Zetasize from Malvern. In some embodiments, the size of the NLC refers to the Z-average particle size.

[0192] This NLC has an average particle size (i.e., number-average particle size) of 1 μm or less. The average particle size (i.e., number-average particle size) of this NLC is approximately 900 nm or less, approximately 800 nm or less, approximately 700 nm or less, approximately 600 nm or less, approximately 500 nm or less, approximately 400 nm or less, 300 nm or less, 200 nm or less, 100 nm or less, or 80 nm or less, for example, approximately 50 nm to approximately 900 nm, approximately 50 nm to approximately 800 nm, approximately 50 nm to approximately 700 nm, approximately 50 nm to approximately 600 nm, approximately 50 nm to It is particularly preferable that the particle size is approximately 500 nm, approximately 50 nm to approximately 400 nm, approximately 50 nm to approximately 300 nm, approximately 50 nm to approximately 200 nm, approximately 50 nm to approximately 175 nm, approximately 50 nm to approximately 150 nm, approximately 50 nm to approximately 125 nm, approximately 50 nm to approximately 100 nm, approximately 50 nm to approximately 80 nm, approximately 40 nm to approximately 80 nm, approximately 20 nm to approximately 80 nm, approximately 40 nm to approximately 80 nm, or approximately 40 nm to approximately 60 nm. Those skilled in the art will understand that NLC is composed of NLC particles. This average particle size refers to the average particle size of each particle constituting the NLC. The average particle size of the NLC particles is typically about 40 nm, 60 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, or 200 nm.

[0193] In some embodiments, the average particle size of the NLC particles is approximately 20 nm to 200 nm, approximately 20 nm to 150 nm, approximately 20 nm to 110 nm, approximately 20 nm to 80 nm, approximately 20 nm to 70 nm, and approximately 20 nm to 60 nm.

[0194] In some embodiments, the average particle size of the NLC particles is approximately 50 nm to 200 nm, approximately 50 nm to 150 nm, approximately 50 nm to 110 nm, approximately 50 nm to 80 nm, approximately 50 nm to 70 nm, and approximately 50 nm to 60 nm.

[0195] In some embodiments, the average particle size of the NLC particles is approximately 40 nm to approximately 80 nm or approximately 40 nm to approximately 60 nm.

[0196] An exemplary NLC of the present invention can be filtered with at least a 0.45 μm filter. In exemplary embodiments, the NLC can be filtered with a 0.20 or 0.22 μm filter.

[0197] B. Stability

[0198] The exemplary NLCs provided herein are stable and offer ease of use, manufacturability, transportability, and storage. The physiological and chemical properties of these NLCs (including, but not limited to, size) are maintained over long periods at a variety of temperatures and under various conditions.

[0199] The evolution of particle size as a function of time provides information about colloidal stability. An exemplary stable NLC composition is one in which its particles maintain substantially the same z-mean particle size over a period of time (e.g., 30 days or 7 days) at various temperatures (typically but not limited to 37°C, 25°C, or 5°C). Maintaining substantially the same z-mean particle size means that the particles remain within 20%, 15%, 10%, or 5% of their original particle size over 30 days. Particularly stable NLC compositions are those in which their particles maintain substantially the same z-mean particle size over 30 days at 4°C, 25°C, or 37°C.

[0200] The stability of this NLC can be measured by methods well known to those skilled in the art. In some embodiments, stability is confirmed visually. Visual inspections include particulate matter inspection, flocculence inspection, or aggregate inspection. Typically, colloidal stability is determined by the particle size of this NLC, such as by measuring the z-mean particle size, and is expressed, optionally, as a change in size over time, a change in size at various temperatures, or a change in size under specific conditions. In some embodiments, stability is determined by evaluating the increase in particle size. In some embodiments, stability is determined by measuring the polydispersity index (PDI) using, for example, dynamic light scattering (DLS). In other embodiments, stability is determined by measuring the zeta potential using DLS.

[0201] In some embodiments, the increase in the Z-mean particle size of the NLC is less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 12%, less than 10%, less than 7%, less than 5%, less than 3%, and less than 1% over the assay period.

[0202] In some embodiments, the polyvariance index of the NLC is maintained at approximately 0.5, approximately 0.4, approximately 0.3, approximately 0.2, approximately 0.1, or approximately 0.1 to approximately 0.5, approximately 0.1 to approximately 0.4, approximately 0.1 to approximately 0.3, approximately 0.1 to approximately 0.2, approximately 0.2 to approximately 0.4, or approximately 0.2 to approximately 0.3. In some preferred embodiments, the polyvariance index is greater than 0.1, greater than 0.15, or greater than 0.2.

[0203] The exemplary NLC-based compositions of the present invention are stable at 25°C for more than 6 months (e.g., maintaining substantially the same z-average particle size).

[0204] IV. Bioactive Drugs

[0205] In some exemplary embodiments, the formulations of the present invention are mixed or formulated with one or more bioactive agents to deliver a bioactive agent. The term “bioactive agent” as used herein means any substance delivered by the formulations of this disclosure, including, but not limited to, polymers, peptides, proteins, peptide mimes, nucleic acids, oligonucleotides, deoxyribonucleotides, plasmid DNA, circular DNA, linear DNA, single-stranded DNA, modified DNA, antisense DNA, ribonucleotides, mRNA, chemically modified RNA, non-coding RNA, miRNA, siRNA, tRNA, ribosomal RNA, RNA ribozymes, replicon RNA, RNA aptamers, DNA aptamers, double-stranded RNA, nucleotide-substituted RNA, inosine-containing RNA, adjuvants, for example, TLR agonists (e.g., TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR7 agonists, TLR8 agonists, and TLR9 agonists), Rig-I agonists, saponins, sugars, sugar polymers, complex carbohydrates, whole virus particles, virus-like particles, virus fragments, and cell fragments. Non-limiting and exemplary adjuvants include double-stranded RNA, RIBOXXOL, Poly(I:C), and Hiltonol® (PolyICLC). Hiltonol® (PolyICLC) is a synthetic complex of carboxymethylcellulose, polyinosinate-polycytidylic acid double-stranded RNA, and poly-L-lysine. RIBOXXOL is an annealed 50 bp double-stranded RNA (Riboxx). Any bioactive agent that can be safely delivered to cells can be mixed with the NLC of the present invention. When a negatively charged molecule is delivered, in some embodiments, the cationic NLC surface can interact with the negatively charged bioactive agent to tether the molecule to the NLC.

[0206] Examples of negatively charged molecules used as bioactive agents include, for example, peptide-containing antigens, nucleic acid molecules encoding one or more peptide-containing antigens (e.g., RNA or DNA), negatively charged polysaccharides, negatively charged small molecules, and negatively charged immunoadjuvants. Examples of negatively charged immunoadjuvants include immunostimulatory oligonucleotides (e.g., CpG oligonucleotides), single-stranded RNA, and small molecule immune potentiators (SMIPs). Examples of negatively charged small molecules include phosphonic acids and fluorophosphonic acids.

[0207] Most recent adjuvants, such as alam, are Th2-biased. In some embodiments, there is a need for Th1-biasing adjuvants for vaccines targeting cancer and infectious diseases (e.g., tuberculosis, several viral diseases), as well as allergies. In this regard, as described herein, the inventors have presented formulations that promote Th1 bias for TLR3 agonists and the like. Such formulations promote IFNγ production, downregulate IL-5, and are suitable for a variety of applications where Th1 bias is desired.

[0208] One or more bioactive agents may be bound to the formulation of the present invention. As will be understood by those skilled in the art, various combinations of bioactive agents may be bound to the formulation, including, but are not limited to, multiple RNAs, multiple DNAs, one or more sequenced RNAs and one or more proteins, one or more DNAs and one or more proteins, and one or more RNAs and one or more DNAs. In some embodiments, one bioactive agent may be present in the oily core of the NLC, while the other bioactive agent may be bound to the surface of the NLC. For example, nucleic acids may be bound to the NLC surface, while biologically active small molecules may be present in the oily core of the NLC.

[0209] In exemplary embodiments, the negatively charged bioactive agent forms a complex with the NLC by binding to a cationic surface. This binding between the negatively charged bioactive agent and the NLC surface may be a non-covalent interaction or a reversible covalent interaction.

[0210] In another embodiment, a hydrophobic bioactive agent, such as a Toll-like receptor ligand (e.g., TLR4 ligand), can be incorporated into the oily core or at the interface of the NLC particles.

[0211] A.RNA molecule

[0212] In embodiments where the bioactive agent is an RNA molecule, the RNA molecule may encode various types of proteins, including, but are not limited to, antigens, antibodies, toxins, growth factors, cytokines, and hormones. The RNA molecules used herein may also be non-coding RNAs, including, but are not limited to, siRNA, miRNA, CRISPR guide RNA, ribozyme RNA, hairpins, RNA aptamers, RNA agonists, and immunomodulatory RNAs.

[0213] In an exemplary embodiment, the negatively charged RNA molecule forms a complex with the NLC by binding to a cationic surface. This binding between the RNA molecule and the NLC surface may be a non-covalent interaction or a reversible covalent interaction.

[0214] In exemplary embodiments, the bioactive agent is a self-replicating RNA molecule. Self-replicating RNA molecules are well known in the art and can be generated by using replication elements derived from viruses (e.g., alphaviruses, flaviviruses, picornaviruses) to replace viral structural proteins with nucleotide sequences encoding the target protein. Self-replicating RNA molecules are typically (+) chain molecules that can be translated directly after delivery to cells. This translation prepares RNA-dependent RNA polymerase, which then produces both antisense and sense transcripts from the delivered RNA. That is, the delivered RNA results in the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, may achieve insight expression of the encoded antigen by translation of themselves, or they may transcribe further transcripts having the same sense chain as the delivered RNA, and achieve insight expression of the antigen by translation of these transcripts. As a result of this entire series of transcriptions, the number of introduced replicon RNAs is amplified, thereby making the encoded antigen the main polypeptide product of the cell.

[0215] An advantage is that self-replicating RNA molecules utilize the cell's translation mechanism, leading to a significant increase in encoded gene products (such as proteins or antigens), which can then be accumulated within the cell or secreted. Self-replicating RNA molecules may induce apoptosis in transfected cells by stimulating Toll-like receptors (TLRs) 3, TLR7, and TLR8, as well as non-TLR pathways (e.g., RIG-I, MD-5), through their RNA replication and amplification products, as well as their translation products.

[0216] The self-replicating RNA may contain at least one or more genes selected from the group consisting of, for example, viral replicases, viral proteases, viral helicases, and other non-structural viral proteins, and may also contain cis-active replication sequences at its 5′ and 3′ ends, and optionally a heterologous sequence encoding the amino acid sequence of interest (e.g., the antigen of interest). The self-replicating RNA may also contain a subgenome promoter that directs the expression of the heterologous sequence. If necessary, this heterologous sequence (e.g., the antigen of interest) may be in-frame fused to another coding region of the self-replicating RNA, with or without the inclusion of a ribosome skipping peptide sequence, or further / or under the control of an intra-sequence ribosome entry site (IRES).

[0217] In one embodiment, the self-replicating RNA molecule is not encapsulated in a virus-like particle. The self-replicating RNA molecule of the present invention can be designed so as not to induce the production of infectious virus particles. This can be achieved, for example, by removing one or more viral genes within the self-replicating RNA that encode structural proteins necessary for the production of virus particles. For example, if the self-replicating RNA molecule is based on alphaviruses such as Sindbis virus (SIN), Semliki forest virus, and Venezuelan encephalitis virus (VEE), one or more genes encoding viral structural proteins such as capsid (C) glycoprotein and / or envelope (E) glycoprotein can be removed.

[0218] If necessary, the self-replicating RNA molecules of the present invention may also be designed to induce the production of attenuated or toxic infectious viral particles, or to produce viral particles capable of causing a single secondary infection.

[0219] One suitable system for achieving such self-replication is the use of alphavirus-based replicas. Alphaviruses include a range of genetically, structurally, and serologically related arthropod-borne viruses of the Togaviridae family. Thirty-one species are classified into the genus Alphavirus, including, for example, Sindobis virus, Semryqui forest virus, Ross River virus, Chikungunya virus, and Venezuelan horse encephalitis virus. Therefore, the self-replicating RNA of the present invention may incorporate RNA replicases derived from Semryqui forest virus (SFV), Sindobis virus (SIN), Venezuelan horse encephalitis virus (VEE), Ross River virus (RRV), Eastern horse encephalitis virus, Chikungunya virus, or other viruses belonging to the genus Alphavirus.

[0220] Alphavirus-based "replicon" expression vectors can be used in this invention. Several types of replicon vectors, including DNA, RNA, and recombinant replicon particles, may be used. Such replicaconvectors include, for example, Sindbis virus (Xiong et al. (1989) Science 243:1188-1191; Dubensky et al., (1996) J. Virol. 70:508-519; Hariharan et al. (1998) J. Virol. 72:950-958; Polo et al. (1999) PNAS 96:4598-4603), Semlik Forest virus (Liljestrom (1991) Bio / Technology 9:1356-1361; Berglund et al. (1998) Nat. Biotech. 16:562-565), and Venezuelan encephalitis virus (Pushko et al. (1997) Virology These are derived from alphaviruses, including 239:389-401). While alphavirus-derived replicons generally share many similar overall characteristics (e.g., structure, replication), individual alphaviruses may exhibit several unique and specific properties (e.g., interferon susceptibility, and disease profile). Therefore, chimeric alphavirus replicons derived from a wide range of viridae can also be useful.

[0221] Alphavirus-based RNA replicons are typically (+)-strand RNAs, which, upon delivery to cells, result in the translation of a replicase (or replicase transcriptase). This replicase is translated as a polyprotein, which then self-cleaves to form a replication complex, which generates a genomic (-)-strand copy of the (+)-strand delivery RNA. Transcription of this (-)-strand transcript itself can yield further (+)-strand parental RNA copies, as well as subgenomic transcripts encoding antigens. That is, translation of the subgenomic transcripts leads to insightful expression of the antigen by infected cells. Suitable alphavirus replicons can utilize replicases derived from Sindbis virus, Semliki forest virus, Eastern equine encephalitis virus, Venezuelan horse encephalitis virus, and others.

[0222] RNA replicons may include RNA genomes obtained from picornaviruses, togaviruses (e.g., alphaviruses such as Sindbis virus, Semlik Forest virus, Venezuelan encephalitis virus, or Ross River virus), flaviviruses (e.g., yellow fever virus), coronaviruses, and paramyxoviruses, modified by substituting one or more structural protein genes with selected heterologous nucleic acid sequences encoding the target product.

[0223] In some embodiments, the replicon encodes (i) an RNA-dependent RNA polymerase capable of transcribing RNA from the replicon, and (ii) an antigen. This polymerase may be, for example, an alphaviral replicase containing one or more of the alphaviral proteins nsP1, nsP2, nsP3, and nsP4. While the natural alphaviral genome encodes structural virion proteins in addition to non-structural replicase polyproteins, it is preferable that the replicon does not encode alphaviral structural proteins. That is, the replicon can produce copies of its own genomic RNA in cells, but not RNA-containing virions. The inability to produce these virions means that, unlike wild-type alphavirus, the preferred replicon cannot persist itself in an infectious form. The alphaviral structural proteins necessary for persistence in wild-type viruses are not present in the preferred replicon, and instead, a gene encoding the target antigen is present, such that the subgenome transcript encodes the antigen rather than a structural alphaviral virion protein.

[0224] A replicon useful in the present invention may have, for example, two open reading frames. In one example, the first (5′) open reading frame encodes a replicase; and the second (3′) open reading frame encodes an antigen. In some embodiments, this RNA may have additional (e.g., downstream) open reading frames to encode additional antigens or accessory polypeptides, for example.

[0225] Replicons can often enhance in vivo translation of RNA by, for example, having a 5′ cap (e.g., 7-methylguanosine). In some embodiments, the 5′ sequence of the replicon may need to be selected to ensure compatibility with the encoded replicase.

[0226] The replicon may have a 3′ poly(A) tail. The replicon may contain a poly(A) polymerase recognition sequence (e.g., AAUAAA) near the 3′ end.

[0227] Replicons can have various lengths, but are typically between 5,000 and 25,000 nucleotides long, for example, 8,000 to 15,000 nucleotides or 9,000 to 12,000 nucleotides long.

[0228] It is preferable that the replicon can be produced by in vitro transcription (IVT). The IVT can utilize a (cDNA) template that has been prepared and grown in plasmid form in bacteria, or prepared by synthesis (e.g., by gene synthesis and / or polymerase chain reaction (PCR)). For example, the replicon can be transcribed from the template DNA using a DNA-dependent RNA polymerase (such as bacteriophage T7 RNA polymerase, bacteriophage T3 RNA polymerase, or SP6 RNA polymerase). If necessary, appropriate cap formation and poly-A addition reactions can also be utilized (however, the poly-A of the replicon is usually encoded in the template DNA). These RNA polymerases may have stringent requirements for the transcribed 5′ nucleotide, and in some embodiments, these requirements must be compatible with the requirements of the encoded replicase so that the RNA transcribed by IVT can function efficiently as a substrate for the replicase that encodes it. Specific examples include Sindbisvirus-based plasmids (pSINs) such as pSINCP, which are described, for example, in U.S. Patents 5,814,482 and 6,015,686, and International Publications 97 / 38087, 99 / 18226, and 02 / 26209. Construction of such replicons is generally described in U.S. Patents 5,814,482 and 6,015,686.

[0229] In other embodiments, the self-replicating RNA molecule is derived from or based on a virus other than an alphavirus, preferably a positive-stranded RNA virus, picornavirus, flavivirus, rubivirus, pestivirus, hepacivirus, calicivirus, or coronavirus. Suitable wild-type alphavirus sequences are well known and can be obtained from sequence depositaries such as the United States Cell Culture Lineage Preservation Service (Rockville, Maryland).Suitable alphaviruses include Aura virus (ATCC VR-368), Beval virus (ATCC VR-600, ATCC VR-1240), Cabassou virus (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCC VR-1241), Eastern Equine Encephalomyelitis virus (ATCC VR-65, ATCC VR-1242), Fort Morgan virus (ATCC VR-924), Geta virus (ATCC VR-369, ATCC VR-1243), Kyzylagach virus (ATCC VR-927), Mayaro virus (ATCC VR-66), Mayaro virus (ATCC VR-1277), and Middleburg virus (ATCC VR-1277). VR-370), Mukambo virus (ATCC VR-580, ATCC VR-1244), Ndumu virus (ATCC VR-371), Pixna virus (ATCC VR-372, ATCC VR-1245), Ross River virus (ATCC VR-373, ATCC VR-1246), Semliki Forest virus (ATCC VR-67, ATCC VR-1247), Sindbis virus (ATCC VR-68, ATCC VR-1248), Tonate virus (ATCC VR-925), Trinity virus (ATCC VR-469), Una virus (ATCC VR-374), Venezuelan equine encephalomyelitis virus (ATCC Examples include VR-69, ATCC VR-923, ATCC VR-1250, ATCC VR-1249, ATCC VR-532), Western equine encephalomyelitis virus (ATCC VR-70, ATCC VR-1251, ATCC VR-622, ATCC VR-1252), Whataroa virus (ATCC VR-926), and Y-62-33 virus (ATCC VR-375).

[0230] In other embodiments, the self-replicating RNA molecule is derived from or based on a replicating virus (e.g., an oncolytic virus). Oncolytic viruses preferentially infect and lyse (destroy) cancer cells. When infected cancer cells are destroyed, new infectious viral particles or virions are released that can infect and destroy further cancer cells. That is, oncolytic viruses not only cause the direct destruction of cancer cells but also stimulate the host's anti-cancer immune response. In some embodiments, the oncolytic virus may encode tumor-related or virus-related antigens, neoantigens, and / or peptides. Suitable oncolytic viruses are known in the art and can be obtained from sequence depositaries such as the United States Cell Culture Lineage Preservation Service (Rockville, Maryland). Representative examples of suitable oncolytic viruses include, but are not limited to, poxviruses, adenoviruses, adeno-associated viruses, reoviruses, retroviruses, seneca viruses, measles viruses, herpes simplex viruses, Newcastle disease viruses (NDV), varicella stomatitis viruses (VSV), mumps viruses, influenza viruses, parvoviruses, human hantaviruses, myxoma viruses, cytomegaloviruses (CMV), lentiviruses, coxsackieviruses, echoviruses, seneca-valley viruses, Sindbisviruses, JX-594, p53-expressing viruses, ONYX-15, Delta24, telomelysin, telomelysin-GFP, and vaccinia, as well as recombinant mutants thereof. In some embodiments, the oncolytic viruses are genetically modified with respect to tumor selectivity. In other embodiments, the oncolytic viruses are naturally occurring. Examples of naturally occurring oncolytic viruses include, but are not limited to, reoviruses and seneca viruses.

[0231] The self-replicating RNA molecules of the present invention, prepared using modified nucleotides, are typically larger than other types of RNA (e.g., mRNA). Typically, the self-replicating RNA molecules of the present invention have at least about 3 kb. For example, the self-replicating RNA may have at least about 4 kb, at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, at least about 11 kb, at least about 12 kb, or more than 12 kb. In certain examples, the self-replicating RNA is about 4 kb to about 12 kb, about 5 kb to about 12 kb, about 6 kb to about 12 kb, about 7 kb to about 12 kb, about 8 kb to about 12 kb, about 9 kb to about 12 kb, about 10 kb ~ about 12kb, about 11kb - about 12kb, about 5kb - about 11kb, about 5kb - about 10kb, about 5kb - about 9kb, about 5kb - about 8kb, about 5kb - about 7kb, about 5kb - about 6kb, about 6kb The ranges are approximately 12kb, 6kb to 11kb, 6kb to 10kb, 6kb to 9kb, 6kb to 8kb, 6kb to 7kb, 7kb to 11kb, 7kb to 10kb, 7kb to 9kb, 7kb to 8kb, 8kb to 11kb, 8kb to 10kb, 8kb to 9kb, 9kb to 11kb, or 10kb to 11kb.

[0232] The self-replicating RNA molecule of the present invention may contain one or more types of modified nucleotides (e.g., pseudouridine, N6-methyladenosine, 5-methylcytidine, 5-methyluridine).

[0233] The self-replicating RNA molecule may encode a single heterologous polypeptide antigen, or, if desired, two or more heterologous polypeptide antigens whose sequences are linked together (e.g., linked in series) in such a way that they retain their uniqueness when expressed as amino acid sequences. The heterologous polypeptides generated from the self-replicating RNA may be generated as fusion polypeptides, or they may be manipulated to become separate polypeptide sequences or peptide sequences.

[0234] The self-replicating RNA of the present invention may encode one or more polypeptides. These polypeptides may consist of binding proteins, enzymes, cytokines, chemokines, hormones, or other functional proteins. Alternatively, these polypeptides may consist of antigens containing a set of epitopes, preferably epitopes capable of eliciting a helper T cell response or a cytotoxic T cell response or both.

[0235] The self-replicating RNA molecules described herein may be manipulated to express multiple nucleotide sequences from two or more open reading frames, thereby enabling the co-expression of proteins, such as two or more antibody sequences or two or more antigens and cytokines or other immunomodulators, thereby enhancing the development of an immune response. Such self-replicating RNA molecules may be particularly useful, for example, in producing bivalent or higher vaccines by simultaneously generating various gene products (e.g., proteins), such as two different single-chain antibody sequences, heavy-chain and light-chain antibody sequences, or multiple antigens.

[0236] The self-replicating RNA molecules of the present invention can be prepared by any preferred method. Several preferred methods for generating RNA molecules containing modified nucleotides are known in the art. For example, self-replicating RNA molecules containing modified nucleotides can be prepared by transcribing (e.g., in vitro transcription) the DNA encoding the self-replicating RNA molecule using a preferred DNA-dependent RNA polymerase, such as T7 phage RNA polymerase, SP6 phage RNA polymerase, T3 phage RNA polymerase, or variants of these polymerases, which allows for the efficient incorporation of modified nucleotides into the RNA molecule. The transcription reaction involves nucleotides and modified nucleotides, as well as other components that support the activity of the selected polymerase, such as preferred buffers and preferred salts. By manipulating the incorporation of nucleic acid analogs into the self-replicating RNA, it may be possible, for example, to alter the stability of the RNA molecule, increase its resistance to ribonucleases, establish replication after introduction into a suitable host cell ("infectivity" of RNA), and / or induce or reduce innate and adaptive immune responses.

[0237] Self-replicating RNA molecules of the present invention can be produced using preferred synthetic methods alone or in combination with one or more other methods (e.g., recombinant DNA techniques or recombinant RNA techniques). Preferred de novo synthetic methods are well known in the art and can be adapted for specific applications. Exemplary methods include, for example, the β-cyanoethyl phosphoramidite method of chemical synthesis using preferred protecting groups such as CEM; and the nucleoside H-phosphonic acid method. These chemical reactions can be performed or adapted for use with commercially available automated nucleic acid synthesizers. Additional preferred synthetic methods are described in Uhlmann et al. (1990) Chem Rev 90:544-84 and Goodchild J (1990) Bioconjugate Chem 1: 165. Nucleic acid synthesis can also be performed using preferred recombinant methods conventionally known in the art, such as cloning, processing, and / or expression of polynucleotides and gene products encoded by such polynucleotides. DNA shuffling by random fragmentation and reconstruction of gene fragments and synthetic polynucleotides by PCR are examples of known techniques that can be used for the design and manipulation of polynucleotide sequences. By altering nucleic acids and the proteins they encode using site-directed mutagenesis, it is possible to, for example, insert new restriction enzyme recognition sites, change glycosylation patterns, alter codon preferences, generate splice variants, or introduce mutations. Preferred methods for the transcription, translation, and expression of nucleic acid sequences are conventionally known in the art.

[0238] The presence and / or content of one or more modified nucleotides in a self-replicating RNA molecule can be confirmed by any preferred method. For example, the self-replicating RNA can be digested to a monophosphate (e.g., using nuclease P1), dephosphorylated (e.g., using a suitable phosphatase such as CIAP), and the resulting nucleoside can be analyzed by reverse-phase HPLC.

[0239] If desired, the self-replicating RNA molecule of the present invention may contain one or more modified nucleotides such that its immunomodulatory activity after introduction, i.e., after entry into a host cell (e.g., a human cell), is reduced compared to a corresponding self-replicating RNA molecule that does not contain modified nucleotides.

[0240] If necessary, self-replicating RNA molecules can be screened or analyzed using various in vitro or in vivo testing methods known to those skilled in the art to confirm their therapeutic and preventive properties. For example, vaccines containing self-replicating RNA molecules can be tested for their effects on inducing proliferation or effector function in specific lymphocyte types of interest (e.g., B cells, T cells, T cell lines, and T cell clones). For instance, spleen cells derived from immunized mice can be isolated to test the ability of cytotoxic T lymphocytes to lyse target autologous cells containing self-replicating RNA molecules encoding polypeptide antigens. Furthermore, helper T cell differentiation can be analyzed by proliferation measurements or ELISA measurements of cytokine production of TH1 (IL-2 and IFN-γ) and / or TH2 (IL-4 and IL-5), or directly in CD4-positive T cells by cytoplasmic cytokine staining and flow cytometry after antigen stimulation.

[0241] Self-replicating RNA molecules encoding polypeptide antigens can also be investigated for their ability to induce humoral immune responses, which can be demonstrated, for example, by inducing the production of antibodies specific to the target antigen in B cells. These assays can be performed, for example, using peripheral B lymphocytes derived from immunized individuals. Such assay methods are known to those skilled in the art. Other assays that can be used to characterize the self-replicating RNA molecules of the present invention may include the detection of the expression of the encoded antigen by target cells. For example, FACS can be used to detect antigen expression on the cell surface or intracellularly. Another advantage of choosing FACS is that it can be screened for differences in expression levels, as lower expression may be preferable. Other preferred methods for identifying cells expressing a specific antigen include panning using monoclonal antibodies on a plate or capture using magnetic beads coated with monoclonal antibodies.

[0242] B.DNA molecule

[0243] In embodiments where the bioactive agent is a DNA molecule, the DNA molecule may encode various types of proteins, including but not limited to antigens, antibodies, toxins, growth factors, cytokines, and hormones. Examples of DNA include, but are not limited to, plasmid DNA, circular DNA, linear DNA, single-stranded DNA, modified DNA, antisense DNA, and aptamer DNA.

[0244] C. Antigen

[0245] The bioactive agents described herein may be nucleic acid molecules (e.g., DNA or RNA) encoding antigens. Suitable antigens include, but are not limited to, bacterial antigens, viral antigens, fungal antigens, protozoan antigens, plant antigens, cancer antigens, or combinations thereof. Antigens may be, for example, those involved in or derived from allergies, cancer, infectious diseases, or autoimmune diseases.

[0246] An antigen may be any target epitope, target molecule (including biomolecules), target molecule complex (including molecular complexes containing biomolecules), target subcellular assembly, target cell, or target tissue against which the induction or enhancement of an immune response in a target is desired. The term antigen often refers to the polypeptide antigen of interest. In some embodiments, the antigen may be an infectious pathogen and / or epitope, biomolecule, cell, or tissue associated with infection, cancer, autoimmune disease, allergy, asthma, or any other condition for which stimulation of an antigen-specific immune response would be desirable or beneficial, or may be derived from such pathogen or tissue, or may be immunologically cross-reactive with such pathogen.

[0247] In certain embodiments, antigens derived from at least one infectious pathogen, such as bacteria, viruses, or fungi, are intended, for example: Actinobacterium, e.g., Mycobacterium tuberculosis or Mycobacterium leprae or another Mycobacterium; Bacteria, e.g., members of the genera Escherichia coli, Salmonella, Neisseria, Borrelia, Chlamydia, Clostridium, or Bordetella; Viruses, e.g., herpes simplex virus, human immunodeficiency virus (HIV such as HIV-1 or HIV-2), influenza virus, parainfluenza virus, measles virus, mumps virus, rubella virus, coronavirus (such as SARS or MERS), rotavirus, norovirus Viruses, picornaviruses (such as poliovirus, enterovirus, or coxsacchie virus), zoonotic pathogens such as feline immunodeficiency virus (FIV), cytomegalovirus, varicella-zoster virus, hepatitis viruses, Epstein-Barr virus (EBV), flaviviruses (such as dengue virus, Japanese encephalitis virus, yellow fever virus, Zika virus, Poissant virus, or tick-borne encephalitis virus), henipaviruses Viruses (such as Hendra virus or Nipah virus), Bunyaviruses (such as Hantavirus or Rift Valley hemorrhagic fever virus), Arenaviruses (such as Lassa virus, Junin virus, Machupo virus, or Guanalitovirus), Filoviruses (such as Ebola virus or Marburg virus), Lyssaviruses (such as rabies virus), Respiratory Syncytial Virus, Human Papillomavirus (HPV), and Cytomegalovirus; Fungi, e.g., Aspergillus, Blastomyces, Coccidioides, and Pneumocystis; or yeasts, e.g., Candida albicans, Candida glabrata, Candida krusei, Candida lusitaniae, Candida tropicalis, and Candida paraprosis (C.Candida species such as parapsilosis; parasites, such as protozoa, such as Plasmodium species, such as Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale; or Acanthamoeba, Entamoeba histolytica, Angiolongillus, Schistosoma mansonii, Schistosoma haematobium, Schistosoma japonicum, Cryptosporidium, Hookworms, Entamoeba histolytica, Entamoeba coli, Entamoeba dispar Other parasites such as *Dispar*, *Entamoeba hartmanni*, *Entamoeba polecki*, *Wuchereria bancrofti*, *Giardia*, *Toxoplasma gondii*, and one or more of the *Leishmania* genera. In certain embodiments, the antigen may be obtained from or related to antigens involved in tuberculosis, influenza, amoebiasis, HIV, hepatitis, or leishmaniasis.

[0248] In some embodiments, the antigen is an influenza-related antigen. In some embodiments, the antigen is an influenza-causing antigen. In some embodiments, the antigen is derived from an influenza-causing virus. In one embodiment, the antigen comprises hemagglutinin (HA) derived from H5N1. In one embodiment, the antigen comprises neuraminidase derived from H5N1.

[0249] For example, in one embodiment, the antigen is derived from a Borrelia species, and the antigen may be a nucleic acid, a pathogen-derived antigen, or an antigenic preparation, a recombinant protein or peptide, or a chimeric fusion protein. One such antigen is OspA. OspA may be a fully matured protein (Lipo-OspA) in which lipids have been added by its biosynthesis in the host cell, or it may be a non-lipidized derivative. Such non-lipidized derivatives include a non-lipidized NS1-OspA fusion protein having the first 81 N-terminal amino acids of the non-structural protein (NS1) of the influenza virus and the complete OspA protein, and separately, MDP-OspA is a non-lipidized form of OspA having three additional N-terminal amino acids.

[0250] In one embodiment, the antigen is HIV-1 (e.g., tat, nef, gp120, or gp160), human herpesvirus (e.g., gD or its derivatives, or pre-initial proteins such as ICP27 derived from HSV1 or HSV2), cytomegalovirus (e.g., human cytomegalovirus, e.g., gB or its derivatives), rotavirus (including attenuated live viruses), Epstein-Barr virus (e.g., gp350 or its derivatives), varicella-zoster virus (e.g., gpl, II, and IE63), or hepatitis B virus (e.g., hepatitis B virus). Hepatitis viruses such as hepatitis surface antigens or their derivatives, hepatitis A virus, hepatitis C virus, and hepatitis E virus, or other viral pathogens, such as paramyxoviruses: respiratory syncytial viruses (e.g., F protein and G protein or their derivatives), parainfluenza virus, measles virus, mumps virus, human papillomavirus (e.g., HPV6, HPV11, HPV16, HPV18, etc.), flaviviruses (e.g., dengue virus, Japanese encephalitis virus, yellow fever virus, Zika virus, Poswanan virus) Antigens are derived from viruses such as the virus, tick-borne encephalitis virus, or influenza virus (whole particle live or inactivated virus, split influenza virus, virus grown in eggs or MDCK cells, or whole particle influenza virosom (described in Gluck, Vaccine, 1992, 10, 915-920), or purified or recombinant proteins thereof, such as HA protein, NP protein, NA protein, PB1 protein, PB2 protein, PA protein, NS1 protein, or M protein, or combinations thereof).

[0251] In another embodiment, the antigen includes Neisseria species, including Neisseria gonorrhoeae and Neisseria meningitidis (e.g., capsular polysaccharides and their conjugates, transferrin-binding proteins, lactoferrin-binding proteins, PilC, adhesins); Streptococcus pyogenes (e.g., M protein or fragments thereof, C5A protease, lipoteichoic acid); Agalactiae, Streptococcus mutans; H. ducreyi; Moraxella species, including Moraxella catarrhalis, also known as Branhamella catarrhalis (e.g., high and low molecular weight adhesins and invasins); Bordetella pertussis (B. Bordetella species including Bordetella parapertussis (e.g., pertussis toxin, pertussis toxin or its derivatives, filamentous hemagglutinin, adenylyl cyclase, pili), Bordetella parapertussis, and Bordetella bronchiseptica; Mycobacterium species including Mycobacterium tuberculosis (e.g., ESAT6, antigen 85A, 85B, or 85C), Mycobacterium bovis, Mycobacterium leprae, Mycobacterium avium, Mycobacterium paratuberculosis, and Mycobacterium smegmatis; Legionella species including Legionella pneumophila; enterotoxic Escherichia coli (E.g., Mycobacterium tuberculosis) (e.g., ESAT6, antigen 85A, 85B, or 85C), Mycobacterium bovis, Mycobacterium leprae, Mycobacterium avium, Mycobacterium paratuberculosis, and Mycobacterium smegmatis; Legionella species including Legionella pneumophila; enterotoxic Escherichia coli (E.g., enterotoxic E. coli). Species of the genus Escherichia coli, including E. coli (e.g., colonization factors, heat-labile toxins or their derivatives, heat-stable toxins or their derivatives), enterohemorrhagic E. coli, enteropathogenic E. coli (e.g., Shiga toxin-like toxins or their derivatives); species of the genus Vibrio, including Vibrio cholera (e.g., cholera toxin or its derivatives); species of Shigella, including Shigella sonnei, Shigella dysenteriae, and Shigella flexnerii; Yersinia enterocolitica (Y.Yersinia species including enterocolitica (e.g., Yop protein), plague bacillus (Y. pestis), and Mycobacter pseudotuberculosis (Y. pseudotuberculosis); Campylobacter species including Campylobacter jejuni (e.g., toxins, adhesin and invasin) and Campylobacter coli; Salmonella species including Salmonella typhi, Salmonella paratyphi, Salmonella choleraesuis, and Salmonella enteritidis; Listeria species including Listeria monocytogenes; Helicobacter species including Helicobacter pylori (e.g., urease, catalase, vacuolated lethal toxin); Pseudomonas species including Pseudomonas aeruginosa; Staphylococcus aureus (S. Staphylococcus species, including Staphylococcus aureus and Staphylococcus epidermidis; Enterococcus species, including Streptococcus fecalis and Enterococcus faecium; Clostridium species, including Clostridium tetani (e.g., tetanus toxin and its derivatives), Clostridium botulinum (e.g., botulinum toxin and its derivatives), and Clostridium difficile (e.g., Clostridium toxin A or toxin B and its derivatives); Bacillus species, including Bacillus anthracis (e.g., botulinum toxin and its derivatives); Corynebacterium species, including Corynebacterium diphtheriae (e.g., diphtheria toxin and its derivatives); Borrelia burgdorferi (B. Borrelia species including Borrelia burgdorferi (e.g., OspA, OspC, DbpA, DbpB), Borrelia garinii (e.g., OspA, OspC, DbpA, DbpB), Borrelia afzelii (e.g., OspA, OspC, DbpA, DbpB), Borrelia andersonii (e.g., OspA, OspC, DbpA, DbpB), and Borrelia hermsii; Ehrlichia equii (E.Ehrlichia species, including the pathogen of human granulocytic ehrlichiosis (equ) and other Ehrlichia species; Rickettsia species, including Rickettsia rickettsii; Chlamydia species, including Chlamydia trachomatis (e.g., MOMP, heparin-binding protein), Chlamydia pneumoniae (e.g., MOMP, heparin-binding protein), and Chlamydia psittaci; Leptospira species, including Leptospira interrogans; Treponema pallidum (e.g., rare outer membrane protein), Treponema denticola, and Treponema hyodienteriae (T. Antigens derived from one or more pathogenic microorganisms, such as Treponema species (including Hyodysenteriae) or other pathogenic microorganisms.

[0252] In another embodiment, the antigen is one or more parasites (e.g., Ohn, DT and Petri, WA, Markell and Voge's Medical Parasitology-9) th Ed., 2006, WB Saunders, Philadelphia; Bowman, DD, Georgis' Parasitology for Veterinarians-8 thAntigens derived from (see Ed., 2002, WB Saunders, Philadelphia), for example, Plasmodium species including Plasmodium falciparum; Toxoplasma species including Toxoplasma gondii (e.g., SAG2, SAG3, Tg34); Entoamoeba species including Entamoeba histolytica; Babesia species including Babesia microti; Trypanosoma species including Trypanosoma cruzi; Giardia species including Giardia lamblia; Leishmania spp. including Leishmania major; Pneumocystis species including Pneumocystis carinii; Trichomonas vaginalis (T.Trichomonas species, including vaginalis; or helminths capable of infecting mammals, e.g., (i) nematode infections (e.g., but not limited to pinworms (Enterobius vermicularis), roundworms (Ascaris lumbricoides), Trichuris trichuria, hookworms (Necator americanus), hookworms (Ancylostoma duodenale), Wuchereria bancrofti, Brugia malayi, Onchocerca volvulus, Dracanculus medinensis, Trichinella spiralis, and Strongyloides stercoralis); (ii) trematode infections (e.g., but not limited to Schistosoma mansoni) (iii) Tapeworm infections (e.g., but not limited to Taenia saginata and Taenia solium). In one embodiment, the antigen is derived from a species of the genus Schistosoma, such as Schistosoma mansonii, Schistosoma haematobium, and / or Schistosoma japonicum, or from a yeast such as a Candida species including Candida albicans, or a Cryptococcus species including Cryptococcus neoformans.

[0253] Other specific antigens are derived from Mycobacterium tuberculosis (M. tuberculosis), such as Th Ra12, Tb H9, Tb Ra35, Tb38-1, Erd14, DPV, MTI, MSL, mTTC2, and hTCC1 (International Publication No. 99 / 51748). Proteins of Mycobacterium tuberculosis (M. tuberculosis) also include fusion proteins and their variants in which at least two, three, four or more polypeptides of Mycobacterium tuberculosis (M. tuberculosis) are fused to larger proteins. Specific fusions include Ra12-TbH9-Ra35, Erd14-DPV-MTI, DPV-MTI-MSL, Erd14DPV-MTI-MSL-mTCC2, Erd14-DPV-MTI-MSL, DPV-MTI-MSL-mTCC2, and TbH9-DPV-MTI (International Publication No. 99151748). Other antigens that may be used include antigens, antigen combinations, and fusion proteins described in U.S. Patent Application Publication No. 2010 / 0129391 and International Publication No. 2008 / 124647. In one exemplary embodiment, the fusion protein is ID93. In one exemplary embodiment, the fusion protein is ID91 (SEQ ID NO: 1).

[0254] The fusion protein ID91 contains a fusion of four Mtb proteins: Rv3619 (pathogenicity factor, EsX family, SEQ ID NO: 2), Rv2389 (produced under hypoxic conditions, reuscitation factor D, SEQ ID NO: 3), Rv3478 (member of the PE / PPE family, SEQ ID NO: 4), and Rv1886 (Ag85A, secretory / membrane protein; mycolyltransferase, SEQ ID NO: 5) (Figure 29). The Mtb antigens contained in ID91 were prioritized based on the lack of human sequence homology and IFN-g secretion by human PBMCs from PPD+ donors (not PPD-donors) after antigen stimulation, ensuring immunogenicity in the human population (Bertholet et al., J Immunol. 181(11):7948-57 (2008)). ID91 protein (GLA-SE), combined with the synthetic Toll-like receptor 4 (TLR4) agonist glucopyranosyllipid adjuvant in a stable emulsion form, exhibits protection against Mtb H37Rv four weeks after a single dose in a preclinical mouse model (Orr et al., J Immunol. 193(6):2911-18 (2014)). This subunit vaccine demonstrated a robust TH1 response (IFN-γ, TNF, and IL-2) to ID91 (ibid.).

[0255] In some embodiments, ID91 may include restriction enzymes well known to those skilled in the art. Examples of such restriction enzymes include, but are not limited to, Ndel, Kpnl, BamHI, EcoRI, and / or HindIII. See, for example, vector pET29 in Figure 38A and SEQ ID NOs. 6 and 12, and vector pET28 in Figure 38B.

[0256] Other specific antigens are derived from the Chlamydia genus, including, for example, high molecular weight protein (HWMP) (International Publication No. 99 / 17741), ORF3 (EP366412), and putative membrane protein (Pmp). Other Chlamydia-derived antigens can be selected from the group described in International Publication No. 99128475. Certain antigens may be derived from Streptococcus species, including, for example, antigens from Streptococcus pneumoniae (e.g., capsular polysaccharides and their complexes, PsaA, PspA, streptoricin, choline-binding proteins), as well as the protein antigen pneumolysin (Biochem Biophys Acta, 1989, 67, 1007; Rubins et al., Microbial Pathogenesis, 25, 337-342), and its detoxified variants (International Publication No. 90 / 06951; International Publication No. 99 / 03884). Other bacterial vaccines include antigens derived from Haemophilus species, such as Haemophilus influenzae type B antigens (e.g., PRP and its complexes), unclassifiable Haemophilus influenzae antigens, such as OMP26, high molecular weight adhesins, P5, P6, protein D, and lipoprotein D, as well as fimbrin and fimbrin-derived peptides (U.S. Patent No. 5,843,464) or multicopy variants or fusion proteins.

[0257] Other specific antigens are antigens derived from hepatitis B. Derivatives of hepatitis B surface antigens are well known in the art and include, in particular, the PreS1 antigen, PreS2 antigen, and S antigen described in European patent applications EP-A414374;EP-A-0304578 and EP198474.

[0258] In other embodiments, the antigens are derived from human papillomavirus (HPV) (such as HPV6 or HPV11) thought to be involved in genital warts, and from HPV viruses (such as HPV16 or HPV18) thought to be involved in cervical cancer. Specific antigens include L1 particles or capsomeres, as well as fusion proteins containing one or more antigens selected from the E6, E7, L1, and L2 proteins of HPV6 and HPV11. Certain forms of fusion proteins include L2E7 disclosed in International Publication No. 96 / 26277, and protein D(1 / 3)-E7 disclosed in GB9717953.5 (PCT / EP98 / 05285). Additional possible antigens include HPV16 antigen, HPV18 antigen, HPV33 antigen, and HPV58 antigen. For example, L1 antigen monomers or L2 antigen monomers, or L1 antigens or L2 antigens appearing collectively as virus-like particles (VLPs), or L1 monoproteins appearing alone in VLP structures or capsomere structures. Such antigens, virus-like particles, and capsomeres are publicly known. See, for example, International Publication Nos. 94 / 00152, 94 / 20137, WO94 / 05792, and WO93 / 02184.

[0259] In other embodiments, the antigen is a fusion protein. The fusion protein may be included alone or as a fusion protein such as E7, E2, or F5; certain embodiments include a VLP containing an L1E7 fusion protein (International Publication No. 96 / 11272). Certain HPV16 antigens include an initial protein E6 or F7 and a protein D carrier fused thereto to form an HPV16-derived protein D-E6 fusion or protein D-E7 fusion, or a combination thereof; or include a combination of E6 or E7 and L2 (International Publication No. 96 / 26277). Alternatively, E6 and E7, which are initial proteins of HPV16 or HPV18, may exist as single molecules (e.g., a protein D-E6 / E7 fusion). The composition may optionally contain one or both of the E6 and E7 proteins on the entire surface of HPV18, for example, in the form of a protein D-E6 fusion protein, a protein D-E7 fusion protein, or a protein D E6 / E7 fusion protein. In addition, the composition may contain antigens derived from other HPV strains, for example, antigens derived from HPV31 strain or HPV33 strain.

[0260] The antigen may be derived from a parasite that causes malaria. For example, antigens derived from Plasmodia falciparum include RTS,S and TRAP. RTS is a hybrid protein composed of virtually the entire C-terminal portion of the perisporozoite (CS) protein of Plasmodia falciparum, linked to the surface (S) antigen of the hepatitis B virus via four amino acids of the preS2 protein of the hepatitis B surface antigen. Its complete structure is disclosed in international patent application PCT / EP92 / 02591, which was published as international publication 93 / 10152 claiming priority under UK patent application 9124390.7. When expressed in yeast, RTS is produced as lipoprotein particles, and when co-expressed with the HBV-derived S antigen, mixed particles known as RTS,S are produced.

[0261] The TRAP antigen is described in international patent application PCT / GB89 / 00895, published as international publication 90 / 01496. One embodiment of the present invention is a malaria vaccine, the antigenic preparation comprising a combination of RTS,S antigen and TRAP antigen. Other malaria parasite antigens that could be candidate components of a multistage malaria vaccine include MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2, Sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP, SALSA, PfEXP1, Pfs25, Pfs28, PFS27125, Pfs16, Pfs48 / 45, Pfs230 of Plasmodium falciparum, and analogs thereof contained in Plasmodium falciparum.

[0262] In one embodiment, the antigen is a cancer cell-derived antigen that may be useful in the treatment of cancer by immunotherapy. For example, the antigen may be a tumor rejection antigen, such as a tumor rejection antigen for prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, kidney cancer, or melanoma cancer. Exemplary cancer-derived antigens or cancer cell-derived antigens include MAGE1, MAGE3, and MAGE4, ​​or other MAGE antigens such as those disclosed in WO99 / 40188, PRAME, BAGE, Lage (also known as NY Eos1), SAGE and HAGE (International Publication No. 99 / 53061), or GAGE ​​(Robbins and Kawakami, 1996 Current Opinions in Immunology 8, pp. 628-636; Van den Eynde et al., International Journal of Clinical & Laboratory Research (1997 & 1998); Correale et al. (1997), Journal of the National Cancer Institute 89, p. 293). These cancer antigens, in non-limiting examples, are expressed in a wide range of tumor types, including melanoma, lung cancer, sarcoma, and bladder cancer. See, for example, U.S. Patent No. 6,544,518.

[0263] Other tumor-specific antigens include, but are not limited to, tumor-specific or tumor-associated gangliosides such as GM2 and GM3, or their complexes with carrier proteins; or autopeptide hormones such as full-length gonadotropin-releasing hormone (GnRH, International Publication No. 95 / 20600), a 10-amino acid short peptide useful in many cancer treatments. In another embodiment, prostate antigens such as prostate-specific antigen (PSA), PAP, PSCA (e.g., Proc. Nat. Acad. Sci. USA 95(4) 1735-1740 1998), PSMA are used, or in one embodiment, a prostate antigen known as Prostase is used (e.g., Nelson, et al., Proc. Natl. Acad. Sci. USA (1999) 96: 3114-3119; Ferguson, et al. Proc. Natl. Acad. Sci. USA 1999. 96, (3114-3119; International Publication No. 98 / 12302; U.S. Publication No. 5,955,306; International Publication No. 98 / 20117; U.S. Publication Nos. 5,840,871 and 5,786,148; International Publication No. 00 / 04149). Other prostate-specific antigens are known from International Publication No. 98 / 137418 and International Publication No. / 004149. Another prostate-specific antigen is STEAP (PNAS 96 14523 14528 7-12 1999).

[0264] Other tumor-related antigens useful in connection with the present invention include Plu-1 (J Biol. Chem 274 (22) 15633-15645, 1999), HASH-1, HasH-2, Cripto (Salomon et al Bioessays 199, 21:61-70, U.S. Patent No. 5,654,140), and Criptin (U.S. Patent No. 5,981,215). Furthermore, antigens particularly relevant to vaccines in cancer treatment include tyrosinase and survivorbin.

[0265] In other embodiments, agents used in the compositions of the present invention include respiratory disease-related antigens, such as respiratory diseases caused by or exacerbated by bacterial infections (e.g., pneumococcal infections), for the prevention and treatment of conditions such as chronic obstructive pulmonary disease (COPD). Physiologically, COPD is defined by the presence of irreversible or partially reversible airway obstruction in patients with chronic bronchitis and / or emphysema (Am J Respir Crit Care Med. 1995 Nov;152(5 Pt 2):S77-121). Exacerbations of COPD are often caused by bacterial (e.g., pneumococcal) infections (Clin Microbiol Rev. 2001 Apr;14(2):336-63).

[0266] D. Nucleic acids encoding antibodies

[0267] The bioactive agents described herein (e.g., RNA) may encode antibodies and / or antigen-binding fragments of antibodies, functionally linked to one or more expression regulatory regions as desired, so that upon delivery to a target, the production of antibodies or antigen-binding fragments occurs in that target. In some embodiments, the bioactive agent may contain heavy and light chain coding sequences in a single open reading frame. In other embodiments, the NLC of the present invention may contain two bioactive agents, one encoding the heavy chain and the other encoding the light chain. In other embodiments, the bioactive agent may contain coding sequences for heavy chain variable regions and light chain variable regions linked by flexible short polypeptide sequences so that the expressed biomolecule binds to the target antigen. In some specific embodiments, the produced antibodies can induce an immune response in an individual.

[0268] E.RNA interference

[0269] In some embodiments, the bioactive polynucleotide bound to the NLC is a non-coding RNA, such as an RNA interference (RNAi) polynucleotide. RNAi molecules are capable of inducing RNA interference through interaction with the RNA interference pathway mechanism in mammalian cells, thereby sequence-specifically degrading or inhibiting the translation of messenger RNA (mRNA) transcripts of transgenes. Two main types of RNAi polynucleotides are small or short interfering RNA (siRNA) and microRNA (miRNA). RNAi polynucleotides can be selected from the group including siRNA, microRNA, double-stranded RNA (dsRNA), small hairpin RNA (shRNA), and expression cassettes, which encode RNA capable of inducing RNA interference. siRNA typically contains 15 to 50 base pairs, preferably 21 to 25 base pairs, and includes a double-stranded structure having a nucleic acid sequence that is identical (fully complementary) or nearly identical (partially complementary) to the coding sequence in the target gene or RNA expressed within the cell. siRNA may have a dinucleotide as a 3′ overhang. siRNA may consist of two annealed polynucleotides, or it may consist of a single polynucleotide forming a hairpin structure.

[0270] MicroRNAs (miRNAs) are small, non-coding RNA gene products, approximately 22 nucleotides long, that direct the disruption or repression of translation of mRNA targets. If the complementarity between the miRNA and the target mRNA is partial, translation of the target mRNA is repressed. If the complementarity is extensive, the target mRNA is cleaved. For miRNAs, this complex binds to a target site typically located in the 3' untranslated region of the mRNA, but this target site usually shares only partial homology with the miRNA. The "seed region," a sequence of approximately seven consecutive nucleotides located at the 5' end of the miRNA that forms a complete base pair with its target, plays a crucial role in the specificity of the miRNA. When the RISC / miRNA complex binds to mRNA, either repression of protein translation or cleavage and degradation of the mRNA can occur.

[0271] F.CRISPR RNA

[0272] In some embodiments, the NLC formulation targets target genes by containing a synthetic small molecule guide RNA (sgRNA) for CRISPR / Cas9 genome editing. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) is a locus containing multiple short direct repeats present in the genomes of approximately 40% of sequenced bacteria and approximately 90% of sequenced archaea. CRISPR functions as the immune system of prokaryotes in that it confers resistance to exogenous genetic elements such as plasmids and phages. The CRISPR system results in a form of adaptive immunity. Short segments of exogenous DNA, called spacers, are inserted into the genome between CRISPR repeats and act as memories of past exposures. Then, CRISPR spacers are used to recognize and silence exogenous genetic elements in a manner similar to RNAi in eukaryotes. Cas9 is an essential protein component of the type II CRISPR / Cas9 system. It forms a complex with two types of RNA called CRISPR RNA (crRNA) and trans-activated crRNA (tracrRNA) to form an active endonuclease, which protects host cells by silencing exogenous genetic elements within invading phages or plasmids.

[0273] RNA-inducible endonucleases based on the CRISPR / Cas9 system have been used for genome editing in eukaryotes. In some embodiments of the present invention, the bioactive agent is an RNA encoding sgRNA and / or Cas9 endonuclease. In some embodiments, the RNA comprises one or more polynucleotides encoding Cas9 and two guide RNAs, where the first guide RNA contains a spacer sequence complementary to the 5' double-strand break (DSB) segment and the second guide RNA contains a spacer sequence complementary to the 3' DSB segment. Both guide RNAs may be prepared as single-molecule guide RNAs (including tracrRNA and crRNA), or one or both may be prepared as bi-molecule guide RNAs containing crRNA and tracrRNA, which are molecules separated and not bound to each other.

[0274] G. polypeptide

[0275] In some embodiments, the one or more bioactive agents are polypeptides. The polypeptide may be a full-length protein or a fragment thereof. In some embodiments, the polypeptide is a peptide. In some embodiments, the polypeptide is a fusion protein. In some specific embodiments, the fusion protein may induce an immune response after administration to an organism. In some embodiments, the polypeptide is an antigen, as described in more detail above. The polypeptide may be prepared by any preferred method known to those skilled in the art, such as recombinant expression.

[0276] H. Small molecules

[0277] In some embodiments, the disclosure generally relates to an NLC composition in which the one or more bioactive agents are small molecules or therapeutic agents for drug delivery. The tightness of the binding between the drug molecule and the NLC may be influenced by the physicochemical properties of the drug, the type and concentration of the surfactant, the type of lipid, and the manufacturing method. In some embodiments, a small molecule drug is encapsulated by the NLC, which is made possible by a liquid lipid phase component of an oily core that provides high drug solubility (Beloqui, A., et al. Nanomedicine 2016; 12(1): 143-161).

[0278] The NLC compositions provided herein may be suitable for drug delivery via various routes of administration. These routes of administration include, but are not limited to, dermal, transdermal, oral, intranasal, intrapulmonary, or ophthalmological administration.

[0279] I. Hormones

[0280] In some embodiments, the one or more bioactive agents conjugated to the NLC are polynucleotides or polypeptides encoding a hormone or hormone analog. In some embodiments, the NLC comprises a lipid complexed with a hormone. The hormone may be selected from the group including human growth hormone, adrenocorticotropic hormone, gonadotropin-releasing hormone, oxytocin, leutinizing-hormone-releasing-hormone, follicle-stimulating hormone, insulin, insulin-like growth factor, leptin, parathyroid hormone, thyroid-stimulating hormone, or several combinations thereof. In some embodiments, the NLC formulation comprises a hormone or hormone analog in combination with a small molecule therapeutic compound as described above.

[0281] J. Adjuvant

[0282] In some embodiments, the NLC is for vaccine delivery, and one or more of the bioactive agents are adjuvants, or the NLC compositions provided herein may be administered co-administered with an adjuvant. As used herein, the term adjuvant refers to a substance that enhances or enhances an immune response. This immune response may be, for example, an antigen-specific immune response to an exogenous antigen.

[0283] Many adjuvants contain substances designed to protect antigens from rapid catabolic reactions, such as aluminum hydroxide or mineral oil, and immune response stimulants, such as lipid A (natural or synthetic). Suitable adjuvants are commercially available and include, for example, incomplete / complete Freudian adjuvants (Difco Laboratories, Detroit, Michigan); Merck Adjuvant 65 (Merck and Company, Inc., Loway, New Jersey); AS-2 and its derivatives (SmithKline Beecham, Philadelphia, Pennsylvania); aluminum salts such as CWS, TDM, Leif, aluminum hydroxide gel (Alam), or aluminum phosphate; calcium salts, iron salts, or zinc salts; insoluble suspensions of acylated tyrosine; acylated sugars; cationic or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A, and Quil A. Cytokines such as GM-CSF or interleukin-2, interleukin-7, or interleukin-12 may also be used as adjuvants.

[0284] Certain exemplary compositions utilize adjuvant systems designed primarily to induce a Th1-type immune response. High levels of Th1 cytokines (e.g., IFN-γ, TNF-α, IL-2, and IL-12) tend to favor the induction of a cellular immune response to an administered antigen. In contrast, high levels of Th2 cytokines (e.g., IL-4, IL-5, IL-6, and IL-10) tend to favor the induction of a humoral immune response. After application of compositions such as those provided herein, patients may maintain immune responses including both Th1 and Th2 responses. In exemplary embodiments where the response is exclusively Th1-type, levels of Th1 cytokines increase significantly above levels of Th2 cytokines. These cytokine levels can be readily assessed using standard test methods. For an overview of this cytokine family, see Mossman & Coffman, Ann. Rev. Immunol. 7:145-173 (1989).

[0285] Specific adjuvants used exclusively to induce Th1-type responses include, for example, monophosphoryl lipid A, e.g., 3-de-O-acylated monophosphoryl lipid A (3D-MPLTM), in combination with aluminum salts (US Patents 4,436,727; 4,877,611; 4,866,034; and 4,912,094). CpG-containing oligonucleotides (with unmethylated CpG dinucleotides) also exclusively induce Th1 responses. Such oligonucleotides are well known and are described, for example, in International Publication Nos. 96 / 02555, 99 / 33488, and US Patents 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, in Sato et al., Science 273:352 (1996). Other exemplary adjuvants include saponins such as Quil A or its derivatives, e.g., QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, Massachusetts); estine; digitonin; or saponins from the genus Gypsophila or Chenopodium quinoa. Other exemplary formulations include two or more saponins in combination with the adjuvants of this disclosure, e.g., combinations of at least two from the group consisting of QS21, QS7, Quil A, 0-estine, or digitonin.

[0286] Other examples of adjuvants useful in connection with this disclosure include Toll-like receptor agonists such as TLR1 agonists, TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR6 agonists, TLR7 agonists, TLR8 agonists, TLR7 / 8 agonists, and TLR9 agonists. Still other examples of adjuvants include imiquimod, gardiquimod, reciquimod, and related compounds.

[0287] In other embodiments, the adjuvant is a glucopyranosyllipid A (GLA) adjuvant as described in whole in U.S. Patent No. 8,609,114 or U.S. Patent No. 8,722,064, which are incorporated herein by reference.

[0288] For example, in one embodiment, the TLR4 agonist is a synthetic GLA adjuvant having the structure of the following formula (VII), [ka] (VII) or a pharmaceutically acceptable salt thereof, in the formula, L1, L2, L3, L4, L5, and L6 may be the same or different, and independently be -O-, -NH-, or -(CH2)-; L7, L8, L9, and L 10 They may be the same or different, they may not exist independently, or they may be -C(=O)-; Y1 is an acidic functional group; Y2 and Y3 may be the same or different, and are independently -OH, -SH, or acidic functional groups; Y4 is either -OH or -SH; R1, R3, R5, and R6 may be the same or different, and independently C 8-13 It is alkyl; R2 and R4 may be the same or different, and C may be independent. 6-11 It is alkyl.

[0289] In some embodiments of the synthetic GLA structure, R 1 , R 3 , R 5 and R 6 is C 10 It is alkyl; R 2 and R 4 is a C8 alkyl group. In one embodiment, R 1 , R 3 , R 5 and R 6 is C 11 It is alkyl; R2 and R 4 is a C9 alkyl group.

[0290] For example, in one embodiment, the TLR4 agonist is a synthetic GLA adjuvant having the structure of the following formula (VIII), or a pharmaceutically acceptable salt thereof.

Chemical formula

[0291] In an embodiment of the above GLA structure, R 1 , R 3 , R 5 and R 6 are C 11 ~C 20 alkyl groups; R 2 and R 4 are C 12 ~C 20 alkyl groups. In another specific embodiment, the GLA has the above formula, wherein R 1 , R 3 , R 5 and R 6 are C 11 alkyl groups; R 2 and R 4 are C [[ID=SI]] 13 alkyl groups. In another specific embodiment, the GLA has the above formula, wherein R 1 , R 3 , R 5 and R 6 are C 10 alkyl groups; R 2 and R 4 are C8 alkyl groups.

[0292] In another specific embodiment, the GLA has the above formula, wherein R 1 , R 3 , R 5 and R 6 [[ID=7S]]are C 11 ~C 20 alkyl groups; R 2 and R 4 are C9~C 20 alkyl groups. <00001OS>It is alkyl. In one embodiment, R 1 , R 3 , R 5 and R 6 is C 11 It is alkyl; R 2 and R 4 It is a C9 alkyl group.

[0293] In one embodiment, the TLR4 agonist is a synthetic GLA adjuvant having the structure of formula (IX) below or a pharmaceutically acceptable salt thereof. [ka] (IX)

[0294] In one embodiment of the above GLA structure, R 1 , R 3 , R 5 and R 6 is C 11 ~C 20 It is alkyl; R 2 and R 4 is C9~C 20 It is alkyl. In one embodiment, R 1 , R 3 , R 5 and R 6 is C 11 It is alkyl; R 2 and R 4 It is a C9 alkyl group.

[0295] In one embodiment, the TLR4 agonist is a synthetic GLA adjuvant having the structure of the following formula (X). [ka] (X)

[0296] In one embodiment of the above GLA structure, R 1 , R 3 , R 5 and R 6 is C 11 ~C 20 It is alkyl; R 2 and R4 is C9~C 20 It is alkyl. In one embodiment, R 1 , R 3 , R 5 and R 6 is C 11 It is alkyl; R 2 and R 4 It is a C9 alkyl group.

[0297] In one embodiment, the TLR4 agonist is a synthetic GLA adjuvant having the structure of formula (XI) below or a pharmaceutically acceptable salt thereof. [ka] (XI)

[0298] In one embodiment of the above GLA structure, R 1 , R 3 , R 5 and R 6 is C 11 ~C 20 It is alkyl; R 2 and R 4 is C9~C 20 It is alkyl. In one embodiment, R 1 , R 3 , R 5 and R 6 is C 11 It is alkyl; R 2 and R 4 It is a C9 alkyl group.

[0299] In one embodiment, the TLR4 agonist is a synthetic GLA adjuvant having the following structure or a pharmaceutically acceptable salt thereof. [ka]

[0300] In one embodiment, the TLR4 agonist is a synthetic GLA adjuvant having the following structure or a pharmaceutically acceptable salt thereof. [ka]

[0301] In one embodiment, the TLR4 agonist is a synthetic GLA adjuvant having the following structure or a pharmaceutically acceptable salt thereof. [ka]

[0302] In another embodiment, attenuated lipid A derivatives (ALDs) are incorporated into the compositions described herein. ALDs are lipid A-like molecules modified or constructed to exhibit reduced or different adverse effects of lipid A. These adverse effects include pyrogenicity, localized Schwarzman reactivity, and chicken embryo 50% lethal dose analysis (CELD). 50 Toxicity can be such that it is assessed by ). Useful ALDs in this disclosure include monophosphoryl lipid A (MLA or MPL) and 3-deacylated monophosphoryl lipid A (3D-MLA or 3D-MPL). Since MLA (MPL) and 3D-MLA (3D-MPL) are well known, they do not need to be described in detail herein.

[0303] In the above TLR4 agonist compounds, the overall charge can be determined based on the functional groups within the molecule. For example, the phosphate group can be negatively charged or neutral depending on its ionization state.

[0304] V. Method for preparing exemplary compositions containing a bioactive agent and a nanostructured lipid carrier.

[0305] As provided herein, one method for producing an NLC described herein includes (a) mixing the solid lipid, the liquid lipid, the cationic lipid, and the hydrophobic surfactant (e.g., sorbitan ester) to form an oil phase mixture; (b) mixing the hydrophilic surfactant and water to form an aqueous phase; and (c) mixing the oil phase mixture with the aqueous phase mixture to form the NLC. In some embodiments, a further step includes combining the bioactive agent with the NLC such that the bioactive agent binds to the surface of the NLC by non-covalent or reversible covalent interactions. Such embodiments are preferred when the bioactive agent is negatively charged, such as an RNA molecule or a DNA molecule. The negative charge on the bioactive agent is interacted with the cationic lipid in the NLC to bind the negatively charged bioactive agent to the NLC. In other embodiments where the bioactive agent is hydrophobic, the bioactive agent is mixed with the components of step (a) to form part of the oil phase mixture. In some embodiments, the bioactive agent may be bound to the surface components of the NLC via covalent interactions.

[0306] The formation of an oil phase mixture by mixing the solid phase lipid, the liquid phase lipid, the cationic lipid, and the hydrophobic surfactant (e.g., sorbitan ester) can be achieved, for example, by heating and sonication. The mixing of the oil phase mixture and the aqueous phase mixture can be achieved by various emulsification methods, including, but not limited to, high-shear emulsification and microfluidization.

[0307] VI. Compositions containing nanostructured lipid carriers

[0308] This specification provides formulations, compositions, and pharmaceutical compositions comprising the NLC composition described herein.

[0309] The composition containing this NLC and the bioactive agent may optionally further contain a pharmaceutically acceptable carrier, excipient, or diluent.

[0310] The compositions described herein may be administered to subjects for any purpose of vaccination, therapeutic use, or diagnostic use.

[0311] This specification provides pharmaceutical compositions comprising the compositions of the present disclosure, further combined with pharmaceutically acceptable carriers, excipients, or diluents.

[0312] In particularly preferred embodiments provided herein, the NLC and pharmaceutical compositions provided herein can be filtered through a 0.45 μm filter. In some embodiments, the pharmaceutical compositions can be filtered through a 0.20 μm filter. In some embodiments, the pharmaceutical compositions can be filtered through a 0.22 μm filter.

[0313] In one embodiment, the present invention relates to a pharmaceutical composition comprising a composition containing NLC and a conjugated bioactive agent. Such a composition may be administered to a subject to stimulate an immune response, such as a nonspecific immune response or an antigen-specific immune response, for the purpose of diagnosing, treating, or preventing a disease or other condition, such as an infection by a biological agent.

[0314] In some other embodiments, the pharmaceutical composition is a vaccine composition comprising the composition described herein in combination with a pharmaceutically acceptable carrier, excipient, or diluent. The exemplary carriers are typically nontoxic to the recipient at the doses and concentrations used.

[0315] In some embodiments, the pharmaceutical compositions provided herein are administered to a subject to induce a response in the subject, for example, to induce an immune response in the subject. Typically, a therapeutically effective dose is administered to the subject.

[0316] The term "effective dose" or "therapeutic effective dose" refers to an amount sufficient to achieve, or at least partially achieve, the desired effect, for example, an amount sufficient to induce the desired immune response. An effective dose of NLC or pharmaceutical composition is administered in an "effective regime." The term "effective regime" refers to a combination of the amount and frequency of administration of the composition that is suitable for achieving the desired effect.

[0317] Actual dose levels may be modified so that a particular patient, composition, and mode of administration yield an effective amount that is non-toxic to the patient and effective in achieving the desired response. The selection of dose levels shall be determined based on a variety of pharmacokinetic factors, combined with the specific composition used, the age, sex, weight, condition, overall health, and medical history of the person being treated, as well as factors well known in the medical field.

[0318] In the exemplary therapeutic embodiments provided herein, therapeutic pharmaceutical compositions are administered in doses ranging from approximately 1 μg / kg to approximately 10 mg / kg. As will be apparent to those skilled in the art, the number and frequency of administrations shall be determined based on the subject's response.

[0319] In the exemplary vaccine-based embodiments provided herein, approximately 1 μg to 100 μg of the antigen or 0.1 μg to 10 mg of the nucleic acid encoding the antigen is administered with each dose. The exemplary formulation enables human administration of approximately 0.1 μg, approximately 1 μg, approximately 5 μg, or approximately 10 μg to approximately 500 μg of replicon RNA. The exemplary formulation enables human administration of approximately 5 μg to approximately 20 μg of replicon RNA.

[0320] As will be apparent to those skilled in the art, the number and frequency of administrations shall be determined based on the subject's response. The exemplary formulation exhibits therapeutic effects after only one dose.

[0321] "Pharmacodynamically acceptable carriers" for therapeutic use are well-known in the pharmaceutical field and are described, for example, in Remingtons Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edit. 1985). For example, sterile saline and phosphate-buffered saline at physiological pH may be used. Preservatives, stabilizers, dyes, and even flavorings may be included in the pharmaceutical composition. For example, sodium benzoate, sorbic acid, and p-hydroxybenzoate esters may be added as preservatives (ibid., 1449). Furthermore, antioxidants and suspending agents may be used (ibid.).

[0322] The form of the pharmaceutical composition is not particularly limited, as long as it allows the composition to be administered to a patient. For example, the composition may be in solid form, liquid form, or gaseous form (aerosol). Common routes of administration include, but are not limited to, oral, topical, parenteral, sublingual, buccal, rectal, vaginal, intravenous, intradermal, transdermal, intranasal, intramucosal, intrapulmonary, or subcutaneous routes. As used herein, the term parenteral includes iontophoresis, ultrasound, thermal, transdermal administration, and even subcutaneous injection, intravenous, intramuscular, intrasternal, intracavernosal, subarachnoid, intratraumatic, and intraurethral injections or infusions. In some embodiments, the compositions described herein (including vaccines and pharmaceutical compositions) are administered intradermally by a method selected from iontophoresis, microcavitation, ultrasound, jet injection, or microneedling. In one preferred embodiment, the compositions described herein are administered intradermally using a microneedle device manufactured by NanoPass Technologies Ltd. (Nesgiona, Israel), such as the MicronJet600 (see, for example, U.S. Patents No. 6,533,949 and No. 7,998,119, which are incorporated herein by reference in their entirety, and Yotam, et al., Human vaccines & immunotherapeutics 11(4): 991-997 (2015)).

[0323] The pharmaceutical composition may also be formulated such that the active ingredients contained in the composition become bioavailable immediately after administration to a subject. The composition to be administered to the subject may take the form of one or more dose units; for example, a tablet may be a single dose unit, and a container of one or more aerosol-form compounds of the present invention may hold multiple dose units.

[0324] For oral administration, excipients and / or binders may be included. Examples include sucrose, kaolin, glycerin, starch dextrin, sodium alginate, carboxymethylcellulose, and ethylcellulose. Colorants and / or flavorings may also be included. A coating shell may be used.

[0325] The composition may take the form of a liquid, such as an elixir, syrup, solution, emulsion, or suspension. As two examples, the liquid may be for oral administration or for delivery by injection. When intended for oral administration, the composition may contain one or more of the following: sweeteners, preservatives, colorants, and flavorings. Compositions intended for administration by injection with a needle and syringe or by needleless jet injection may contain one or more of the following: surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0326] Liquid pharmaceutical compositions used herein, whether in liquid form, suspension form, or other similar form, may contain one or more of the following carriers or excipients: sterile diluents such as distilled water for injection, saline solution, preferably physiological saline, Ringer's solution, or isotonic sodium chloride; non-volatile oils such as squalene, squalane, mineral oil, mannide monooleate, or cholesterol; and / or synthetic monoglycerides or synthetic diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents that can act as a solvent or suspension medium; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and tonic modifiers such as sodium chloride or glucose.

[0327] In another embodiment, the compositions of the present disclosure are formulated to be aerosolizable.

[0328] It may be preferable to include other components in the pharmaceutical composition, such as delivery bases, including, but not limited to, aluminum salts, water-in-oil emulsions, biodegradable oil bases, oil-in-water emulsions, biodegradable microcapsules, and liposomes. Examples of additional immunostimulants (coadjuvants) used in such bases are those described above, and may also include N-acetylmuramyl-L-alanine-D-isoglutamine (MDP), glucans, IL-12, GM-CSF, γ-interferon, and IL-12.

[0329] Any suitable carrier known to those skilled in the art may be used in the pharmaceutical compositions of this disclosure, the type of carrier shall be determined based on the mode of administration and whether or not sustained release is intended. For parenteral administration, such as subcutaneous injection, the carrier may include water, saline, alcohol, fat, wax, or buffer. For oral administration, any of the above carriers may be used, or solid carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate may be used. Biodegradable microspheres (e.g., polylactic galactide) may also be used as carriers for the pharmaceutical compositions of the present invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109. In this regard, the microspheres are preferably larger than approximately 25 μm.

[0330] The pharmaceutical composition may contain diluents such as buffers, antioxidants such as ascorbic acid, polypeptides, proteins, amino acids, sugars including glucose, sucrose, or dextrin, chelating agents such as EDTA, glutathione, and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumimskilln is an example of a suitable diluent. For example, the product may be formulated as a lyophilized product using a suitable excipient solution (e.g., sucrose) as a diluent.

[0331] Pharmaceutical compositions may be intended for topical administration, in which case the carrier may appropriately include a solution base, emulsion base, ointment base, or gel base. The base may include, for example, one or more of the following: diluents such as petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, water, and alcohol, as well as emulsifiers and stabilizers. Pharmaceutical compositions for topical administration may also include thickeners. When intended for transdermal administration, the composition may include a transdermal patch or an iontophoresis device. Topical formulations may contain an antigen (e.g., a vaccine composition with GLA as the antigen) or GLA (e.g., an immunoadjuvant composition; GLA is available from Avanti Polar Lipids, Inc. (Alabaster, Alabama); e.g., product number 699800) at a concentration of about 0.1 w / v% to about 10 w / v% (weight per unit volume).

[0332] The composition may be in the form of a suppository or other preparation that dissolves in the rectum and releases the drug for rectal administration. The composition for rectal administration may contain an oily base as a suitable non-irritating excipient. Examples of such bases include, but are not limited to, lanolin, cocoa butter, and polyethylene glycol. In the method of the present invention, the pharmaceutical composition / adjuvant may be administered using an insert, beads, sustained-release formulation, patch, or immediate-release formulation.

[0333] If desired, the NLC may contain physiological salts (such as sodium salts) to adjust the tonicity. For example, about 0.9% (w / v) sodium chloride (NaCl) (physiological saline) may be used. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, magnesium chloride, and calcium chloride. Nonionic tonicity enhancers may also be used to adjust the tonicity. In the compositions of this disclosure, monosaccharides classified as aldoses, such as glucose, mannose, arabinose, and ribose, and monosaccharides classified as ketoses, such as fructose, sorbose, and xylulose, may be used as nonionic tonicity enhancers. Disaccharides such as sucrose, maltose, trehalose, and lactose may also be used. In addition, alditols (acyclic polyhydric alcohols, also known as sugar alcohols), such as glycerol, mannitol, xylitol, and sorbitol, are also useful nonionic tonicity enhancers in the compositions of this disclosure. The nonionic tonicity regulator can be present at a concentration of approximately 0.1% to approximately 10% or approximately 1% to approximately 10%, depending on the nonionic tonicity regulator used. When formulating NLC for parenteral administration, it is preferable to make the osmolality of the NLC composition the same as that of a normal physiological solution in order to prevent post-administration events such as swelling or rapid absorption of the composition after administration.

[0334] If desired, NLC may be formulated with cryoprotective agents including trehalose, sucrose, mannitol, sorbitol, Avicel PH102 (microcrystalline cellulose), Avicel RC591 (a mixture of microcrystalline cellulose and sodium carboxymethylcellulose), Microcelac® (a mixture of lactose and Avicel), or a combination thereof. If desired, NLC may be formulated with preservatives such as Hydrolite 5.

[0335] VII. Stable Emulsions

[0336] In some embodiments, a stable emulsion is provided, which comprises at least one adjuvant. Examples of adjuvants that may be formulated with the stable emulsion include, but are not limited to, TLR3 agonists and Rig-I agonists. Examples of such adjuvants include, but are not limited to, double-stranded RNA, RIBOXXOL, Poly(I:C), and Hiltonol®.

[0337] In some embodiments, the stable emulsion (SE) is an oil-in-water emulsion. In some such embodiments, the oil-in-water emulsion is a squalene emulsion in water. In some embodiments, the emulsion contains an antioxidant such as α-tocopherol (vitamin E, see, e.g., European Patent No. 0382271B1). International Publications 95 / 17210 and 99 / 11241 discuss emulsions based on squalene, α-tocopherol, and TWEEN® 80. International Publication 99 / 12565 discusses improvements to these squalene emulsions by adding sterols to the oil phase.

[0338] International Publication No. 08 / 153541 discusses an oil-in-water emulsion in which the content of various components is typically in the range of 2-10% oil (such as squalene); 0.01-0.1% α-tocopherol, if present; and 0.3-3% surfactant (such as polyoxyethylene sorbitan monooleate or poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene)). The stability of the emulsion can be improved by reducing the oil:surfactant ratio to 1 or less. Span 85 may be present at a level of about 1%. In some cases, it may be advantageous for the vaccine to further contain stabilizers. In one embodiment, the stabilizer is tricaprylin (C 27 H 5OThis may be a triglyceride such as O6 (see, for example, International Publication No. 98 / 56414). In some embodiments, the oil-in-water emulsion contains 0.5% to 5%, 0.5% to 5%, 0.5% to 3%, or 1% to 3% glycerol. In some embodiments, the oil-in-water emulsion contains 0.5% to 5%, 0.5% to 5%, 0.5% to 3%, or 1% to 3% 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Examples of oil-in-water emulsions described herein include, but are not limited to, those described in the examples herein.

[0339] The size of oil droplets present in a stable oil-in-water emulsion is preferably less than 1 μm, measured by photon correlation spectroscopy, and generally within the range of 30 to 600 nm, preferably about 30 to 500 nm in diameter, most preferably about 150 to 500 nm in diameter, and particularly about 150 nm in diameter. In this regard, approximately 80% of the oil droplets should be within the above preferred range, more preferably more than 90%, and most preferably more than 95% of the oil droplets should be within the size range defined above. The content of the various components present in the oil emulsion of the present invention is usually in the range of 2 to 10% oil (such as squalene); if present, in the range of 2 to 10% α-tocopherol; and in the range of 0.3 to 3% surfactant (such as polyoxyethylene sorbitan monooleate). When the ratio of oil to α-tocopherol exceeds 1, a more stable emulsion is obtained, which is preferable. Span85 may be present at a level of about 1%. In some cases, it may be advantageous for the vaccine of the present invention to further contain a stabilizer.

[0340] Methods for producing oil-in-water emulsions are well known to those skilled in the art. Typically, such methods involve mixing the oil phase with a surfactant, such as a PBS / TWEEN80® solution, and then homogenizing it using a homogenizer. For example, a method involving passing the mixture through a syringe needle one, two, or more times would be suitable for homogenizing small amounts of liquid. Similarly, an emulsification step in a microfluidizer (M110S microfluidic machine, maximum input pressure 6 bar (output pressure approximately 850 bar), 2 minutes, up to 50 passes) can be configured to produce smaller or larger amounts of emulsion. This configuration can be achieved by conventional experimental methods, which involve measuring the resulting emulsion until a preparation containing oil droplets of the required diameter is obtained.

[0341] VIII. Methods Using the Compositions Disclosed herein

[0342] A. Treatment drugs

[0343] In some embodiments, the agent is useful for therapeutic purposes. That is, in some embodiments, the composition comprises the NLC provided herein and further comprises a bioactive agent for the treatment of a disease, abnormality, or disorder.

[0344] In some embodiments, the agent is useful for treating or preventing allergies, cancer, infections, autoimmune conditions, or poisoning. In some embodiments, the agent is useful for stimulating, enhancing, and / or modulating immune responses.

[0345] In some embodiments of the disclosed embodiments, the composition comprises a cancer antigen or a nucleic acid encoding a cancer antigen. In some embodiments, the vaccine composition comprises a cancer antigen useful against any cancer characterized by the expression of a tumor-associated antigen, such as HER-2 / neu expression or other cancer-specific or cancer-associated antigens.

[0346] Compositions and methods in certain embodiments of the present disclosure may be used for the prevention or treatment of autoimmune diseases, which include diseases, abnormalities or disorders in which the host or target immune system adversely mediates an immune response targeting “self” tissues, cells, biomolecules (e.g., peptides, polypeptides, proteins, glycoproteins, lipoproteins, proteolips, lipids, glycolipids, nucleic acids such as RNA and DNA, oligosaccharides, polysaccharides, proteoglycans, glycosaminoglycans, etc., as well as other molecular components of the target cells and tissues), or epitopes (e.g., immunologically defined specific recognition structures, such as those recognized by the complementarity-determining region (CDR) of the variable region of an antibody or by the CDR of a T cell receptor).

[0347] In other words, autoimmune diseases are characterized by an abnormal immune response involving cells or antibodies that target normal self-tissues. Autoimmune diseases in mammals can generally be classified into one of two distinct categories: cell-mediated diseases (i.e., T-cell-mediated diseases) or antibody-mediated disorders. Non-exclusive examples of cell-mediated autoimmune diseases include multiple sclerosis, rheumatoid arthritis, Hashimoto's thyroiditis, type 1 diabetes mellitus (juvenile-onset diabetes), and autoimmune uveoretinitis. Antibody-mediated autoimmune disorders include, but are not limited to, myasthenia gravis, systemic lupus erythematosus (or SLE), Graves' disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia, autoimmune asthma, cryoglobulinemia, thrombic thrombocytopenic purpura, primary biliary sclerosis, and pernicious anemia. The associated antigens for systemic lupus erythematosus are small nuclear ribonucleoproteins (snRNPs); the associated antigens for Graves' disease are thyroid-stimulating hormone receptors, thyroglobulin, and other components of thyroid epithelial cells; the associated antigens for pemphigus are cadherinoid pemphigus antigens such as desmoglein 3 and other adhesion molecules; and the associated antigens for thrombic thrombocytopenic purpura are platelet antigens.

[0348] The compositions provided herein may be used to induce protective immunity against tuberculosis and other diseases, and include the use of polypeptides containing at least one immunogenic moiety of one or more Mycobacterium proteins, as well as DNA and RNA molecules encoding such polypeptides. Furthermore, such compounds may be formulated into vaccines and / or pharmaceutical compositions for immunization against Mycobacterium infections.

[0349] In other embodiments, the compositions of the present disclosure include antigens associated with respiratory diseases, such as respiratory diseases caused by or exacerbated by bacterial infections (e.g., pneumococcal infections), for the prevention and treatment of conditions such as chronic obstructive pulmonary disease (COPD).

[0350] In addition to direct in vivo methods, exo-vivo methods may be used, in which cells are collected from a host, modified, and transplanted into the same or a different host animal. It is clear that any of the above compositions for introducing nucleic acid molecules encoding antigens into tissue cells can be used in exo-vivo methods. Protocols for viral, physical, and chemical uptake methods are well known in the art.

[0351] In some embodiments, the compositions of the present disclosure are used to boost or enhance the immune response in a subject. In some such embodiments, the bioactive agent is an adjuvant. Examples of adjuvants include, but are not limited to, TLR agonists (TLR2, TLR3, TLR4, TLR7, TLR8, and TLR9 agonists), Rig-I agonists, saponins, sugars, sugar polymers, complex carbohydrates, whole virus particles, virus-like particles, virus fragments, and cell fragments. Examples of such adjuvants include, but are not limited to, double-stranded RNA, RIBOXXOL, Poly(I:C), and Hiltonol®. In some embodiments, the composition comprises a stable emulsion and / or a nanostructured lipid carrier. In some embodiments, the composition comprises a stable emulsion and / or a nanostructured lipid carrier containing squalene. The inventors have found that squalene-based formulations unexpectedly enhance TLR3 agonists and the like.

[0352] In some preferred embodiments, the compositions of this disclosure are useful for enhancing or inducing an immune response in a host, patient, or cell culture. As used herein, the term “subject” refers to any mammal. A patient may have an infection, cancer such as breast cancer, or an autoimmune disease, or may be normal (i.e., free from detectable disease and / or infection). “Cell culture” is not particularly limited to any preparation containing immune cells or isolated immune system cells (including, but not limited to, T cells, macrophages, monocytes, B cells, and dendritic cells). Such cells can be isolated by any of the various methods well known to those skilled in the art (e.g., Ficol-Highpack density centrifugation). The cells may (though not necessarily) be isolated from a cancer patient and may be reintroduced to the patient after processing.

[0353] B. Vaccines

[0354] In other words, the present disclosure provides compositions that alter the immune response in a host capable of initiating an immune response (i.e., statistically significantly increase or decrease it, for example, compared to a suitable control well known to those skilled in the art). As will be apparent to those skilled in the art, the immune response is not particularly limited as long as it is an active change in the host's immune state, and may include, for example, any changes in the structure or function of one or more tissues, organs, cells, or molecules involved in maintaining and / or regulating the host's immune state. Typically, an immune response can be detected by any of a variety of well-known parameters, including, but not limited to, confirmation in vivo or in vitro of: soluble immunoglobulins or antibodies; soluble mediators such as cytokines, lymphokines, chemokines, hormones, and growth factors, as well as other soluble mediators such as low molecular weight peptides, sugars, nucleotides, and / or lipids; changes in the cellular activation state, confirmed by changes in the functional or structural properties of immune system cells, such as the induction of specific activities including cell proliferation, changes in motility, specific gene expression, or cytolytic behavior; cell differentiation by immune system cells, or the initiation of apoptosis (programmed cell death), including changes in surface antigen expression profiles; or any other assessment criteria that can detect the presence or absence of an immune response.

[0355] It will be readily apparent to those skilled in the art that confirmation of the induction of an immune response by the compositions of this disclosure can be established by any of several well-known immunological testing methods. Such testing methods include, but are not limited to, confirmation in vivo or in vitro of: soluble antibodies; soluble intermediaries such as cytokines, lymphokines, chemokines, hormones, growth factors, and other soluble intermediaries such as low molecular weight peptides, sugars, nucleotides, and / or lipids; changes in the cellular activation state confirmed by changes in the functional or structural properties of immune system cells, e.g., induction of specific activities such as cell proliferation, changes in motility, specific gene expression, or cytolytic behavior; and initiation of cell differentiation or apoptosis (programmed cell death) by immune system cells, including changes in surface antigen expression profiles. Procedures for carrying out these and similar tests are well known and can be found, for example, in Lefkovits' *Immunology Methods Manual: The Comprehensive Sourcebook of Techniques*, 1998; see also *Current Protocols in Immunology*; for example, Weir, *Handbook of Experimental Immunology*, 1986 Blackwell Scientific, Boston, MA; Mishell and Shigii (eds.) *Selected Methods in Cellular Immunology*, 1979 Freeman Publishing, San Francisco, CA; Green and Reed, 1998 *Science* 281:1309, and the references cited therein).

[0356] The detection of antigen-reactive T cell proliferation can be achieved by various known methods. For example, T cell proliferation can be detected by measuring the rate of DNA synthesis, and antigen specificity can be confirmed by adjusting the stimulus to which candidate antigen-reactive T cells are exposed (e.g., antigen-presenting cells pulsed with a specific target antigen or control antigen). T cells stimulated to proliferate show an increase in the rate of DNA synthesis. A typical method for measuring the rate of DNA synthesis is, for example, by pulse-labeling T cell cultures with tritium-labeled thymidine, a nucleoside precursor inserted into the newly synthesized DNA. The amount of inserted tritium-labeled thymidine can be determined using a liquid scintillation spectrophotometer. Other methods for detecting T cell proliferation include measuring increased interleukin-2 (IL-2) production, Ca2+ influx, or the uptake of dyes such as 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium. Alternatively, the synthesis of lymphokines (such as interferon-γ) can be measured, or the relative number of T cells capable of reacting to a specific antigen may be quantified.

[0357] The detection of antigen-specific antibody production can be achieved by assaying a sample (e.g., an immunoglobulin-containing sample such as serum, plasma, or blood) taken from a host treated with the vaccine of this disclosure using an in vitro method such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), equilibrium dialysis, or solid-phase immunoblotting including Western blotting. In each embodiment, the ELISA assay may further include antigen capture and fixation of the target antigen with a solid-phase antigen-specific monoclonal antibody for purposes such as enhancing assay sensitivity. The production of soluble mediators (e.g., cytokines, chemokines, lymphokines, prostaglandins, etc.) can also be readily measured by enzyme-linked immunosorbent assay (ELISA) using methods, apparatus, and reagents readily available from commercial sources (e.g., Sigma, St. Louis, Missouri; see also R&D Systems 2006 Catalog, R&D Systems, Neapolis, Minnesota).

[0358] Any number of other immunological parameters may be monitored using commonly known assays in the art. These assays may include, for example, antibody-dependent cell-mediated cytotoxicity (ADCC) assays, secondary in vitro antibody reactions, immunological cell fluorescence flow analysis of various subpopulations of peripheral blood mononuclear cells or lymphoid mononuclear cells using established marker antigen systems, immunohistochemistry, or other relevant assays. These and other assays can be found, for example, in Rose et al. (Eds.), Manual of Clinical Laboratory Immunolog, 5th Ed., 1997 American Society of Microbiology, Washington, DC.

[0359] Accordingly, the compositions provided herein are intended to be able to induce or enhance in a host at least one immune response selected from Th1 T lymphocyte response, TH2 T lymphocyte response, cytotoxic T lymphocyte (CTL) response, antibody response, cytokine response, lymphokine response, chemokine response, and inflammatory response. In one embodiment, the immune response may include the production of one or more cytokines selected from interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), the production of one or more interleukins selected from IL-1, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, IL-18, and IL-23, the production of one or more chemokines selected from MIP-1α, MIP-1β, Lantes, CCL2, CCL4, CCL5, CXCL1, and CXCL5, and at least one lymphocyte response selected from memory T cell responses, memory B cell responses, effector T cell responses, cytotoxic T cell responses, and effector B cell responses.

[0360] C. Diagnostic drugs

[0361] In some embodiments, the bioactive agent is a diagnostic agent. That is, in some embodiments, the composition comprises the NLC provided herein and further comprises a diagnostic agent useful for diagnosing a disease, abnormality, or disorder.

[0362] In some embodiments, the diagnostic agent is useful for detecting cancer. Compositions and methods for identifying subjects with cancer or suspected to be at risk of developing cancer are known in the art and are described herein. The diagnosis of cancer in subjects with cancer or suspected to be at risk of developing cancer may be achieved by any of the various methods recognized in the art, which can be determined based on a variety of factors including clinical symptoms, stage of cancer, type of cancer, and other factors. Examples of cancer diagnosis include histopathological examination, cytohistochemical examination, immunocytohistochemical and immunopathological examination of patient samples (e.g., blood, skin biopsy, other tissue biopsy, surgical specimen, etc.), PCR testing for a given genetic (e.g., nucleic acid) marker, serological testing for circulating cancer-associated antigens, cells having such antigens, or antibodies having a given specificity, or other methods well known to those skilled in the art.

[0363] In some embodiments, the diagnostic agent is useful for detecting autoimmune diseases. That is, detection of autoantibodies allows for the early detection or recognition of the presence of an autoimmune disease or the risk of developing an autoimmune disease. Based on these findings, various autoantibodies against autoantigens have been discovered, and evaluation of these autoantibodies against autoantigens is being conducted in clinical trials.

[0364] In one embodiment, the diagnostic agent is useful for detecting infectious diseases. Compositions and methods for identifying subjects having an infection with an infectious pathogen as described herein, or subjects suspected to be at risk of developing such an infection, are known in the art.

[0365] For example, the bacterium Mycobacterium tuberculosis causes tuberculosis (TB). That is, in some embodiments, a composition comprising any of the NLCs described herein further comprises an agent for diagnosing tuberculosis.

[0366] In some embodiments, a composition comprising any of the NLCs described herein further comprises a drug for diagnosing malaria.

[0367] Polynucleotides encoding species-specific Plasmodium vivax malaria peptide antigens, which are proteins or protein fragments secreted into the plasma of susceptible mammalian hosts after infection, have been reported in the art, and similarly, monoclonal or polyclonal antibodies against these antigens have also been reported. These peptide antigens, monoclonal antibodies, and / or polyclonal antibodies are used in tests used for the diagnosis of malaria and for determining whether Plasmodium vivax is the species involved in the infection.

[0368] Furthermore, the external domain of recombinant Plasmodium falciparum (3D7) AMA-1 was expressed by a method that generates a high-purity protein that retains the folding and disulfide crosslinking of the native molecule. This recombinant AMA-1 is useful as a diagnostic reagent, for antibody production, and as a vaccine. Similarly, the expression and purification of recombinant Plasmodium falciparum (3D7) MSP-142, which retains the folding and disulfide crosslinking of the native molecule, is also known. This recombinant MSP-142 is useful as a diagnostic reagent, for antibody production, and as a vaccine.

[0369] In some embodiments, a composition comprising any of the NLCs described herein further comprises an agent useful for the diagnosis of leshmaniasis.

[0370] In some embodiments, a composition comprising any of the NLCs described herein further comprises an agent useful for the diagnosis of HIV.

[0371] IX. Methods for inducing an immune response

[0372] This specification provides a method for inducing an immune response in a subject, comprising the step of administering a therapeutically effective amount of a composition described herein to a subject in need thereof, wherein the bioactive agent is a protein antigen or a nucleic acid molecule encoding a protein antigen. In exemplary embodiments, the bioactive agent is an RNA (e.g., mRNA) molecule or DNA molecule encoding a protein antigen. In some embodiments, a method for boosting or enhancing an immune response is provided, wherein the bioactive agent is an adjuvant.

[0373] Typical routes of administration of the therapeutically effective amount of the composition include, but are not limited to, oral, topical, parenteral, sublingual, buccal, rectal, vaginal, intravenous, intradermal, transdermal, nasal, mucosal, or subcutaneous routes. In some exemplary embodiments, the composition is administered intramuscularly, by eye, parenterally, or intrapulmonaryly.

[0374] In exemplary embodiments, the compositions disclosed herein are vaccine compositions used as vaccines. The compositions described herein can be used to induce an immune response (including nonspecific and antigen-specific responses) in a subject. In some embodiments, the immune response includes a systemic immune response. In some embodiments, the immune response includes a mucosal immune response. Inducing an immune response includes stimulating, boosting, or enhancing the immune response.

[0375] The compositions described herein may be used to enhance protective immunity against viruses. Such viruses and viral antigens include, for example, HIV-1 (e.g., tat, nef, gp120 or gp160), human herpesvirus (e.g., gD or its derivatives, or pre-initial proteins such as ICP27 derived from HSV1 or HSV2), cytomegalovirus (in particular human cytomegalovirus, e.g., gB or its derivatives), rotavirus (including attenuated live viruses), Epstein-Barr virus (e.g., gp350 or its derivatives), varicella-zoster virus (e.g., gpl, II and IE63), or hepatitis B virus. For example, hepatitis viruses such as hepatitis B surface antigen or its derivatives, hepatitis A virus, hepatitis C virus, and hepatitis E virus, or other viral pathogens, such as paramyxovirus: respiratory syncytial virus (e.g., F protein and G protein or their derivatives), parainfluenza virus, measles virus, mumps virus, human papillomavirus (e.g., HPV6, HPV11, HPV16, HPV18, etc.), flavivirus (e.g., dengue virus, Japanese encephalitis virus, yellow fever virus, Zika virus, Poswanan virus) Examples include influenza viruses (whole-particle live or inactivated viruses, split influenza viruses, viruses grown in eggs or MDCK cells, or whole-particle influenza virosoms (described in Gluck, Vaccine, 1992, 10, 915-920), or their purified or recombinant proteins, such as HA protein, NP protein, NA protein, or M protein, or combinations thereof).

[0376] The compositions described herein may be used to enhance protective immunity against one or more pathogenic microorganisms, for example, Neisseria species including Neisseria gonorrhoeae and Neisseria meningitidis (e.g., capsular polysaccharides and their conjugates, transferrin-binding proteins, lactoferrin-binding proteins, PilC, adhesins); Streptococcus pyogenes (e.g., M protein or fragments thereof, C5A protease, lipoteichoic acid), Agalactiae, Streptococcus mutans; H. ducreyi; Moraxella species including Moraxella catarrhalis, also known as Branhamella catarrhalis (e.g., high and low molecular weight adhesins and invasins); Bordetella pertussis (B. Bordetella species including Bordetella parapertussis (e.g., pertussis toxin, pertussis toxin or its derivatives, filamentous hemagglutinin, adenylyl cyclase, pili), Bordetella parapertussis, and Bordetella bronchiseptica; Mycobacterium species including Mycobacterium tuberculosis (e.g., ESAT6, antigen 85A, 85B, or 85C), Mycobacterium bovis, Mycobacterium leprae, Mycobacterium avium, Mycobacterium paratuberculosis, and Mycobacterium smegmatis; Legionella species including Legionella pneumophila; enterotoxic Escherichia coli (E.g., Mycobacterium tuberculosis) (e.g., ESAT6, antigen 85A, 85B, or 85C), Mycobacterium bovis, Mycobacterium leprae, Mycobacterium avium, Mycobacterium paratuberculosis, and Mycobacterium smegmatis; Legionella species including Legionella pneumophila; enterotoxic Escherichia coli (E.g., enterotoxic E. coli). Species of the genus Escherichia coli, including *Escherichia coli* (e.g., colonization factors, heat-labile toxins or their derivatives, heat-stable toxins or their derivatives), enterohemorrhagic *Escherichia coli*, enteropathogenic *Escherichia coli* (e.g., Shiga toxin-like toxins or their derivatives); species of the genus *Vibrio*, including *V. cholera* (e.g., cholera toxin or its derivatives); *S. sonnei*, *S. dysenteriae*, *S. flexneri* (S.Shigella species including flexnerii; Yersinia species including Yersinia enterocolitica (e.g., Yop protein), Yersinia pestis, Mycobacter pseudotuberculosis; Campylobacter species including Campylobacter jejuni (e.g., toxins, adhesin and invasin) and Campylobacter coli; Salmonella species including Salmonella typhi, Salmonella paratyphi, Salmonella choleraesuis, Salmonella enteritidis; Listeria species including Listeria monocytogenes; Helicobacter species including Helicobacter pylori (e.g., urease, catalase, vacuolated lethal toxin); Pseudomonas aeruginosa (P. Species of the genus Pseudomonas, including aeruginosa; species of Staphylococcus, including Staphylococcus aureus and Staphylococcus epidermidis; species of Enterococcus, including Streptococcus fecalis and Enterococcus faecium; species of Clostridium, including Clostridium tetani (e.g., tetanus toxin and its derivatives), Clostridium botulinum (e.g., botulinum toxin and its derivatives), and Clostridium difficile (e.g., Clostridium toxin A or toxin B and its derivatives); species of Bacillus, including Bacillus anthracis (e.g., botulinum toxin and its derivatives); and Clostridium diphtheriae (C. Corynebacterium species including diphtheriae (e.g., diphtheria toxin and its derivatives); Borrelia burgdorferi (e.g., OspA, OspC, DbpA, DbpB), Borrelia garinii (e.g., OspA, OspC, DbpA, DbpB), Borrelia afzelii (e.g., OspA, OspC, DbpA, DbpB), Borrelia andersoni (B.Borrelia species including andersonii (e.g., OspA, OspC, DbpA, DbpB), Borrelia hermsii; Ehrlichia species including Ehrlichia equi and the pathogen of human granulocytic ehrlichiosis; Rickettsia species including Rickettsia rickettsii; Chlamydia species including Chlamydia trachomatis (e.g., MOMP, heparin-binding protein), Chlamydia pneumoniae (e.g., MOMP, heparin-binding protein), Chlamydia psittaci; Leptospira species including Leptospira interrogans; Treponema pallidum (T. Pathogenic microorganisms include *Treponema* species (e.g., *Treponema pallidum*, *Treponema denticola*, *Treponema hyodysenteriae*, and other pathogenic microorganisms.

[0377] The compositions described herein may be used to enhance protective immunity against one or more parasites (e.g., Ohn, DT and Petri, WA, Markell and Voge's Medical Parasitology-9). th Ed., 2006, WB Saunders, Philadelphia; Bowman, DD, Georgis' Parasitology for Veterinarians-8 thSee Ed., 2002, WB Saunders, Philadelphia), for example, Plasmodium species including Plasmodium falciparum; Toxoplasma species including Toxoplasma gondii (e.g., SAG2, SAG3, Tg34); Entoamoeba species including Entamoeba histolytica; Babesia species including Babesia microti; Trypanosoma species including Trypanosoma cruzi; Giardia species including Giardia lamblia; Leishmania spp. including Leishmania major; Pneumocystis species including Pneumocystis carinii; Trichomonas vaginalis (T.Trichomonas species, including vaginalis; or helminths capable of infecting mammals, e.g., (i) nematode infections (e.g., but not limited to pinworms (Enterobius vermicularis), roundworms (Ascaris lumbricoides), Trichuris trichuria, hookworms (Necator americanus), hookworms (Ancylostoma duodenale), Wuchereria bancrofti, Brugia malayi, Onchocerca volvulus, Dracanculus medinensis, Trichinella spiralis, and Strongyloides stercoralis); (ii) trematode infections (e.g., but not limited to Schistosoma mansoni) (iii) Tapeworm infections (e.g., but not limited to Taenia saginata and Taenia solium). In one embodiment, the antigen is derived from: species of the genus Schistosoma, Schistosoma mansonii, Schistosoma haematobium, and / or Schistosoma japonicum, or yeast, such as Candida species including Candida albicans; Cryptococcus neoformans (C.Infectious pathogens such as Cryptococcus species (including neoformans), bacteria, viruses, or fungi, including the following: Actinobacterium, for example, Mycobacterium tuberculosis (M. tuberculosis), Mycobacterium leprae (M. Mycobacterium leprae) or other Mycobacterium; Bacteria, e.g., members of the genera Salmonella, Neisseria, Borrelia, Chlamydia or Bordetella; Viruses, e.g., herpes simplex virus, human immunodeficiency virus (HIV), feline immunodeficiency virus (FIV), cytomegalovirus, varicella-zoster virus, hepatitis viruses, Epstein-Barr virus (EBV), Zika virus (ZIKV) respiratory syncytial virus, human papillomavirus (HPV), and cytomegalovirus; HIV, e.g., HIV-1 or HIV-2; Fungi, e.g., Aspergillus, Blastomyces, Coccidioides, and Pneumocystis, or Candida species, e.g., Candida albicans, Candida glabrata, Candida crusae Yeasts including Candida krusei, Candida lusitaniae, Candida tropicalis, and Candida parapsilosis; parasites, such as protozoa, such as Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale (P.Plasmodium species, including ovale; or one or more of other parasites, such as Acanthamoeba, Entamoeba histolytica, Schistosoma, Schistosoma mansonii, Schistosoma haematobium, Schistosoma japonicum, Cryptosporidium species, hookworms, Entamoeba histolytica, Entamoeba coli, Entamoeba dispar, Entamoeba hartmanni, Entamoeba polecki, Wuchereria bancrofti, Giardia, and Leishmania.

[0378] Methods for determining whether the compositions of the present invention can effectively deliver a bioactive agent and / or have the desired effect in a target are known in the art and therefore will not be described in detail herein. In one embodiment, the immune response to an antigen can be determined by monitoring the levels of antigen-specific antibodies (e.g., systemic IgM, IgG (IgG1, IgG2a, etc.), or IgA) in blood samples or from mucous membranes before and after administration. The post-administration cellular immune response can also be monitored by evaluating the function of T cells and B cells after antigen stimulation.

[0379] In another method for evaluating the immunogenicity of a composition or vaccine disclosed herein, in which the nucleic acid molecule (e.g., RNA) encodes a protein antigen, the recombinant protein antigen is expressed for screening of a patient's serum or mucosal secretions by immunoblotting and / or microarray. A positive reaction between the protein and the patient sample indicates that the patient has initiated an immune response to the protein of interest. This method may also be used to identify immunodominant antigens and / or intraepitopes of protein antigens.

[0380] The efficacy of the composition can also be determined in vivo by challenging an appropriate animal model with the target infectious disease pathogen.

[0381] In the embodiments provided herein, the subject is a mammal (for example, animals including livestock (cows, pigs, goats, horses, etc.), pets (cats, dogs, etc.), and rodents (rats, mice, etc.), or humans). In one embodiment, the subject is a human. In another embodiment, the subject is a mammal other than a human. In another embodiment, the mammal other than a human is a dog, a cow, or a horse.

[0382] X. Methods for delivering bioactive agents to cells

[0383] This specification provides a method for delivering a bioactive agent to cells, comprising the step of contacting the cells with a composition described herein. In some embodiments, the bioactive agent is a nucleic acid. In some embodiments, if the cells are present in a subject, the contact between the cells and the composition comprises the step of administering the composition to the subject. Such methods are useful for delivering antigens or nucleic acids encoding antigens to induce an immune response. Such methods are also useful for delivering nucleic acids encoding antibodies, protein agents, small molecule agents, hormones, non-coding RNA molecules, and other bioactive agents for the treatment of diseases and health conditions.

[0384] The methods described herein for delivering bioactive agents to cells may be useful in the treatment of diseases and health conditions, including, but are not limited to, cancers such as meningioma, hepatocellular carcinoma, and pancreatic tumors; allergies; infections including fungal, bacterial, or parasitic diseases; inflammatory diseases including psoriasis and arthritis; atrial-ventricular malformation; autoimmune diseases; and neurological disorders.

[0385] In embodiments of a method for delivering a composition to cells, which includes the step of administering the composition to a target where cells are present, typical routes of administration of a therapeutically effective amount of the composition include, but are not limited to, oral, topical, parenteral, sublingual, buccal, rectal, vaginal, intravenous, intradermal, transdermal, nasal, mucosal, or subcutaneous routes. In preferred embodiments, the administration of the composition is intramuscular, parenteral, or intradermal. In such embodiments, the target is a mammal (e.g., animals including livestock (cows, pigs, goats, horses, etc.), pets (cats, dogs, etc.), and rodents (rats, mice, etc.), or humans). In one embodiment, the target is a human. In another embodiment, the target is a non-human mammal. In another embodiment, the non-human mammal is a dog, a cow, or a horse.

[0386] In some embodiments, multiple delivery modes may be used to produce a larger immune response. For example, the composition may be administered once, twice, three times, or four times. In some embodiments, this one or more administrations may be carried out as part of a so-called "prime-boost" protocol. In some embodiments, this "prime-boost" method includes administration in several stages presenting the same antigen using different vectors or multiple administrations. In some embodiments, more than two administrations may be performed (e.g., three, four, etc.), with two or more boosting administrations following an initial priming administration. When multiple vectors are administered or repeated administrations are performed, each administration may be separated from each other by a period of time, for example, one week, two weeks, three weeks, one month, six weeks, two months, three months, six months, one year, or longer. In some embodiments, the prime-boost method includes an RNA stage and a protein stage. The RNA stage may include, for example, the administration of RNA having a gene encoding an antigen protein, the translation of the RNA into the antigen, and the production of a corresponding antibody in the target. The protein step may include, for example, the direct administration of an antigen in the form of a protein. In some embodiments, a subject is administered (e.g., primed) an oncolytic virus encoding a neoantigen (which may be formulated with or without NLC), followed by administration (e.g., boosted) an NLC containing an RNA construct encoding the neoantigen.

[0387] Intradermal delivery of XI.RNA (using MicronJet600™ if desired)

[0388] MicronJet600® is a small plastic device equipped with three microneedles, each 600 μm (0.6 mm) long. This device can be attached to any standard syringe in place of a standard needle. The microneedles themselves are made from silicon crystals, which are cut in a line and then bonded (bonded) to a polycarbonate base using a biocompatible UV-curing adhesive.

[0389] Intradermal delivery can be performed using a microneedle with a height of 1 mm, or less than 1000 μm, more preferably 500 to 750 μm.

[0390] Microneedle injection devices preferably have multiple needles, and typically have three microneedles.

[0391] The microneedle injection device is oriented "downward" (with the bevel facing downward), meaning the injection device penetrates deeper into the skin and the bevel does not face upward. This ensures reliable injection without leakage. It is preferable that the injection orientation be determined visually or by the mechanical characteristics of the base / adapter.

[0392] Microneedle injections are administered to the superficial dermis and epidermis. This allows for effective expression and immunization.

[0393] The injection depth using microneedles is typically around 100–750 μm, more preferably around 300–400 μm, which differs from conventional needles or other mini-needles or microneedles that typically deliver to deeper skin layers or below the skin.

[0394] The injection angle is preferably about 45° (usually ±20°, more preferably ±10°), which allows for a shallower injection point compared to standard needles and other vertical microneedles.

[0395] This specification provides a system and method for delivering RNA containing rvRNA (replicon RNA) to an animal or human patient (e.g., a subject), the system and method comprising administering the RNA (e.g., rvRNA) to the epidermis or dermis of the skin at a depth of about 100 to about 700 μm from the surface of the skin. The delivery of an effective amount of RNA enables the expression of a protein encoded by the RNA. Such a protein may be an antigen as described herein, or a vaccine component, etc.

[0396] The RNA can be administered using an intradermal delivery device equipped with one or more microneedles and designed for superficial intradermal delivery.

[0397] The RNA may be administered using an intradermal delivery device based on the teachings of U.S. Patent No. 6,533,949 and / or No. 7,998,119, which are incorporated herein by reference in their entirety.

[0398] Any RNA-containing formulations and / or compositions described herein can be administered intradermally by the microneedle devices described herein. Other RNA intradermal delivery devices may also be used, such as intradermal electroporation delivery devices. In some preferred embodiments, RNA delivery induces an immune response in the target.

[0399] XII. Methods to optimize RNA delivery to cells

[0400] This specification provides a method for optimizing RNA delivery to cells, comprising the step of selecting a nitrogen (N):phosphate (P) molar ratio that optimizes the antibody titer produced in a subject including the cells. In exemplary embodiments, the N:P ratio actually used gives findings of RNA-NLC binding and, accordingly, in vitro expression of the RNA-encoded protein. Exemplary N:P molar ratios may be 1 to 200, 1 to 100, preferably 1 to 50, more preferably about 5 to about 50, or about 5 to about 40. In some exemplary embodiments, the N:P molar ratio may be 1 to 15 or 1 to 7.

[0401] XIII. Kits and Products

[0402] In some embodiments, a kit comprising the nanostructured lipid carriers (NLCs) and compositions described herein is also intended, and these NLCs and compositions may be provided in one or more containers. In one embodiment, all components of the composition are present together in a single container. In other embodiments, each component of the composition may be present in two or more containers. In a preferred embodiment, the NLC is provided in one container and the bioactive agent is provided in another container.

[0403] In some embodiments, one vial of the kit contains the NLC provided herein, and a second vial of the kit contains an RNA molecule. In some embodiments, the kit includes a third vial containing an optional component.

[0404] The kit of the present invention may further include instructions for use, as described herein, or instructions for mixing each substance contained in each vial. In some embodiments, the substances in the vials are in a dry or lyophilized state. In some embodiments, the substances in the vials are liquid.

[0405] The container in such a kit embodiment may be any suitable container, vessel, vial, ampoule, tube, cup, box, bottle, flask, jar, dish, well of a single-well instrument, well of a multi-well instrument, reservoir, tank, etc., or other device that can be used to store, preserve and / or transport the compositions disclosed herein and to extract the contents. Typically, such a container may be made of materials suitable for its intended use and the stored contents may be easily recovered. Non-limiting examples of such containers include sealed or resealable tubes and ampoules made of glass and / or plastic, which have rubber dividers or other sealing means suitable for extracting the contents using needles and syringes. Such containers may be made of glass, chemically compatible plastic or resin, for example, or may be made of or coated with a material that allows for efficient extraction of the substance from the container and / or protects the substance from degrading conditions such as ultraviolet light or extremely high or low temperatures, and from the introduction of undesirable contaminants, including contaminating bacteria. Preferably, such containers are sterile or sterilizable and made of a material compatible with any carrier, excipient, solvent, base, etc., which may be used to suspend or dissolve the vaccine compositions and / or immunoadjuvant compositions and / or antigens and / or recombinant expression constructs described herein.

[0406] XIV. Examples of Embodiments Embodiment 1 A composition comprising nanostructured lipid carrier (NLC) particles for delivering a bioactive agent to cells, (a) an oily core containing a mixture of liquid-phase lipids and solid-phase lipids, (b) Cationic component, preferably cationic lipid, (c) Hydrophobic surfactant, preferably sorbitan ester, and (d) Surfactant, preferably hydrophilic surfactant The NLC particles, including the aforementioned NLC particles. Embodiment 2 The composition according to Embodiment 1, wherein the bioactive agent is bound to the NLC particles. Embodiment 3 A composition according to Embodiment 1 or Embodiment 2, which delivers the bioactive agent to the cells. Embodiment 4 The composition according to any one of Embodiments 1 to 3, wherein the hydrophobic surfactant is a sorbitan ester, and the sorbitan ester is present in an amount sufficient to enhance the composition's ability to deliver the bioactive agent to the cells compared to a control composition that does not contain a sorbitan ester. Embodiment 5 The composition according to any one of Embodiments 1 to 4, wherein the bioactive agent is a protein, or the bioactive agent encodes a protein. Embodiment 6 The composition according to any one of Embodiments 1 to 4, wherein the bioactive agent is a protein antigen, or the bioactive agent encodes a protein antigen. Embodiment 7 The composition according to Embodiment 6, wherein the cells are cells within the target, and the composition induces an immune response of the target to the antigen. Embodiment 8 The composition according to Embodiment 6 or Embodiment 7, wherein the antigen is an antigen derived from an infectious pathogen and / or epitope, biomolecule, cell or tissue associated with an infectious disease, cancer or autoimmune disease, or is an antigen that is immunoreactive with such an antigen. Embodiment 9 The composition according to any one of embodiments 6 to 8, wherein the hydrophobic surfactant is a sorbitan ester, and the sorbitan ester is present in an amount sufficient to enhance the ability of the composition to induce an immune response to the antigen compared to a control composition that does not contain a sorbitan ester. Embodiment 10 The composition according to any one of Embodiments 6 to 9, which, when administered to the subject in an effective amount, induces an immune response to the antigen that is equivalent to or greater than the immune response induced when the bioactive agent is administered to the subject without the NLC. Embodiment 11 The composition according to any one of Embodiments 6 to 10, wherein the hydrophobic surfactant is a sorbitan ester, and when administered to the subject in an effective amount, it induces an antibody titer against the antigen at a level higher than that induced when a control composition without a sorbitan ester is administered to the subject. Embodiment 12 The composition according to any one of Embodiments 6 to 11, wherein the hydrophobic surfactant is a sorbitan ester, and induces a neutralizing antibody titer in the subject that is higher than the neutralizing antibody titer induced in the subject by a control composition that does not contain a sorbitan ester. Embodiment 13 The composition according to any one of Embodiments 1 to 12, wherein the biological agent is RNA or DNA. Embodiment 14 The composition according to any one of Embodiments 1 to 12, wherein the biological activator is mRNA. Embodiment 15 The composition according to any one of Embodiments 1 to 12, wherein the bioactive agent is oncolytic viral RNA. Embodiment 16 The composition according to Embodiment 13 or Embodiment 14, wherein the RNA is a replicon. Embodiment 17 The composition according to any one of embodiments 13 to 16, wherein the RNA encodes an antigen. Embodiment 18 The composition according to any one of embodiments 13 to 16, wherein the RNA encodes an antibody. Embodiment 19 The composition according to any one of embodiments 13 to 16, wherein the RNA is a non-coding RNA. Embodiment 20 The composition according to any one of Embodiments 1 to 19, wherein the liquid-phase lipid is metabolizable. Embodiment 21 The composition according to any one of Embodiments 1 to 20, wherein the liquid-phase lipid is a vegetable oil, an animal oil, or an oil produced by synthesis. Embodiment 22 The composition according to any one of Embodiments 1 to 21, wherein the liquid phase lipid is fish oil. Embodiment 23 The composition according to any one of Embodiments 1 to 20, wherein the liquid-phase lipid is tri(caprylic / capric acid) glyceryl, vitamin E, lauroyl polyoxylglyceride, monoacylglycerol, soy lecithin, squalene, or squalane, or a combination thereof. Embodiment 24 The composition according to any one of Embodiments 1 to 21, wherein the liquid-phase lipid is squalene, sunflower oil, soybean oil, olive oil, grape seed oil, squalane, caprylic / capric triglyceride, or a combination thereof. Embodiment 25 The composition according to any one of Embodiments 1 to 21, wherein the liquid-phase lipid is a natural terpenoid or a synthetic terpenoid. Embodiment 26 The composition according to any one of Embodiments 1 to 21, wherein the liquid-phase lipid is squalene. Embodiment 27 The composition according to any one of Embodiments 1 to 26, wherein the solid phase lipid is a glycerolipid. Embodiment 28 The composition according to any one of Embodiments 1 to 26, wherein the solid phase lipid is a microcrystalline triglyceride. Embodiment 29 The composition according to Embodiment 28, wherein the microcrystalline triglyceride is trimiristin. Embodiment 30 The aforementioned cationic components are 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol), dimethyldioctadecylammonium (DDA), 1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP), dipalmitoyl(C16:0)trimethylammoniumpropane (DPTAP), distearoyltrimethylammoniumpropane (DSTAP), and N-[1-(2,3-dioleyl The composition according to any one of Embodiments 1 to 29, wherein the cationic lipid is selected from oxy)propyl-N,N,N-trimethylammonium chloride (DOTMA), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), and 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), and combinations thereof. Embodiment 31 The composition according to Embodiment 30, wherein the cationic lipid is 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP). Embodiment 32 The composition according to any one of Embodiments 1 to 31, wherein the hydrophilic surfactant is polyethylene glycol. Embodiment 33 The composition according to Embodiment 32, wherein the hydrophilic surfactant is polyoxyethylene sorbitan ester. Embodiment 34 The composition according to any one of Embodiments 1 to 33, wherein the mean polydispersity index of the NLC particles is 0.1 to about 0.5. Embodiment 35 The composition according to any one of Embodiments 1 to 33, wherein the average polydispersity index of the NLC particles is about 0.2 to about 0.5. Embodiment 36 The composition according to any one of Embodiments 1 to 33, wherein the mean polydispersity index of the NLC particles is about 0.2 to about 0.4 or about 0.1 to about 0.4. Embodiment 37 The composition according to any one of Embodiments 1 to 33, wherein the mean polydispersity index of the NLC particles is about 0.2 to about 0.3 or about 0.1 to about 0.3. Embodiment 38 The composition according to any one of Embodiments 1 to 37, wherein the z-average particle size of the NLC particles is about 40 nm to about 60 nm. Embodiment 39 The composition according to any one of Embodiments 1 to 37, wherein the z-average particle size of the NLC particles is approximately 20 nm to approximately 200 nm, approximately 20 nm to approximately 150 nm, approximately 20 nm to approximately 100 nm, approximately 20 nm to approximately 80 nm, approximately 20 nm to approximately 60 nm, approximately 40 nm to approximately 200 nm, approximately 40 nm to approximately 150 nm, approximately 40 nm to approximately 100 nm, approximately 40 nm to approximately 80 nm, or approximately 40 nm to approximately 60 nm. Embodiment 40 The composition according to any one of Embodiments 1 to 39, wherein the hydrophobic surfactant is a sorbitan ester having a hydrophilic-lipophilic balance (HLB) value of 1 to 5. Embodiment 41 The composition according to Embodiment 40, wherein the hydrophobic surfactant is a sorbitan ester having an HLB value of 4 to 5. Embodiment 42 The composition according to any one of Embodiments 1 to 40, wherein the hydrophobic surfactant is a sorbitan ester, and the sorbitan ester is a sorbitan monoester. Embodiment 43 The composition according to any one of Embodiments 1 to 40, wherein the hydrophobic surfactant is a sorbitan ester, and the sorbitan ester is sorbitan monostearate. Embodiment 44 The composition according to any one of Embodiments 1 to 40, wherein the hydrophobic surfactant is a sorbitan ester, and the sorbitan ester is a sorbitan monooleate. Embodiment 45 The composition according to any one of Embodiments 1 to 44, wherein the z-average particle size of the NLC particles is about 40 nm to about 80 nm. Embodiment 46 The composition according to any one of Embodiments 1 to 44, wherein the z-average particle size of the NLC particles is about 40 nm to about 60 nm. Embodiment 47 The composition according to any one of Embodiments 1 to 46, wherein the oil-surfactant molar ratio is about 0.5 to about 12, more preferably about 0.5 to about 9. Embodiment 48 The composition according to any one of Embodiments 1 to 46, wherein the oil-surfactant molar ratio is about 0.5 to about 1. Embodiment 49 Hydrophilic surfactant: A composition according to any one of Embodiments 1 to 48, wherein the ratio of cationic components is about 0.2 to about 1.5. Embodiment 50 Hydrophilic surfactant: A composition according to any one of Embodiments 1 to 48, wherein the ratio of cationic components is about 0.2 to about 1. Embodiment 51 A composition according to any one of Embodiments 1 to 50, wherein the RNA loading capacity is at least about 100 μg / mL. Embodiment 52 The composition according to any one of Embodiments 1 to 51, wherein the hydrophobic surfactant is a sorbitan ester, and the sorbitan ester is a sorbitan triester. Embodiment 53 The composition according to any one of Embodiments 1 to 51, wherein the hydrophobic surfactant is a sorbitan ester, and the sorbitan ester is a sorbitan trioleate. Embodiment 54 A composition according to any one of Embodiments 1 to 51, comprising approximately 0.2 w / v% to approximately 40 w / v% liquid-phase lipids, approximately 0.1 w / v% to approximately 10 w / v% solid-phase lipids, approximately 0.2 w / v% to approximately 10 w / v% cationic lipids, approximately 0.25 w / v% to approximately 5 w / v% sorbitan monoester, and approximately 0.5 w / v% to approximately 10 w / v% hydrophilic surfactant. Embodiment 55 A composition according to any one of Embodiments 1 to 51, comprising approximately 2 w / v% to approximately 40 w / v% liquid-phase lipids, approximately 0.1 w / v% to approximately 10 w / v% solid-phase lipids, approximately 0.2 w / v% to approximately 10 w / v% cationic lipids, approximately 0.25 w / v% to approximately 5 w / v% sorbitan monoester, and approximately 0.5 w / v% to approximately 10 w / v% hydrophilic surfactant. Embodiment 56 A composition according to any one of Embodiments 1 to 53, comprising approximately 10 w / v% to approximately 40 w / v% liquid-phase lipids, approximately 1% to approximately 2% solid-phase lipids, approximately 2% to approximately 5% cationic lipids, approximately 3% to approximately 5% sorbitan ester, and approximately 3% to approximately 5% hydrophilic surfactant. Embodiment 57 A composition according to any one of Embodiments 1 to 53, comprising approximately 15 w / v% liquid-phase lipids and approximately 1% solid-phase lipids, or approximately 30 w / v% liquid-phase lipids and approximately 1.8% solid-phase lipids, as well as approximately 3% cationic lipids, approximately 3.7 w / v% sorbitan ester, and approximately 3.7 w / v% hydrophilic surfactant. Embodiment 58 A composition according to any one of Embodiments 1 to 53, comprising approximately 2 w / v% to approximately 6 w / v% liquid-phase lipids, approximately 0.1 w / v% to approximately 1 w / v% solid-phase lipids, approximately 0.2 w / v% to approximately 1 w / v% cationic lipids, approximately 0.25 w / v% to approximately 1 w / v% sorbitan monoester, and approximately 0.5 w / v% to approximately 5 w / v% hydrophilic surfactant. Embodiment 59 A composition according to any one of Embodiments 1 to 53, comprising approximately 3.75 w / v% liquid-phase lipids, approximately 0.25 w / v% solid-phase lipids, approximately 3 w / v% cationic lipids, approximately 3.7 w / v% sorbitan ester, and approximately 3.7 w / v% hydrophilic surfactant. Embodiment 60 A composition according to any one of Embodiments 1 to 53, comprising approximately 0.2 w / v% to approximately 40 w / v% liquid-phase lipids, approximately 0.1 w / v% to approximately 10 w / v% solid-phase lipids, approximately 0.2 w / v% to approximately 10 w / v% cationic lipids, approximately 0.25 w / v% to approximately 5 w / v% sorbitan esters, and approximately 0.2 w / v% to approximately 10 w / v% hydrophilic surfactants. Embodiment 61 A composition according to any one of Embodiments 1 to 53, comprising approximately 0.2 w / v% to approximately 40 w / v% liquid-phase lipids, approximately 0.1 w / v% to approximately 10 w / v% solid-phase lipids, approximately 0.2 w / v% to approximately 10 w / v% cationic lipids, approximately 0.25 w / v% to approximately 15 w / v% sorbitan esters, and approximately 0.2 w / v% to approximately 15 w / v% or approximately 0.5 w / v% to approximately 15 w / v% hydrophilic surfactants. Embodiment 62 A composition according to any one of Embodiments 1 to 53, comprising approximately 2 w / v% to approximately 40 w / v% liquid-phase lipids, approximately 0.1 w / v% to approximately 10 w / v% solid-phase lipids, approximately 0.2 w / v% to approximately 10 w / v% cationic lipids, approximately 0.25 w / v% to approximately 5 w / v% sorbitan esters, and approximately 0.2 w / v% to approximately 10 w / v% hydrophilic surfactants. Embodiment 63 A composition according to any one of Embodiments 1 to 53, comprising approximately 2 w / v% to approximately 6 w / v% liquid-phase lipids, approximately 0.1 w / v% to approximately 1 w / v% solid-phase lipids, approximately 0.2 w / v% to approximately 1 w / v% cationic lipids, approximately 0.25 w / v% to approximately 1 w / v% sorbitan monoester, and approximately 0.2 w / v% to approximately 5 w / v% hydrophilic surfactant. Embodiment 64 A composition according to any one of Embodiments 1 to 53, comprising approximately 4 w / v% liquid-phase lipids, approximately 0.25 w / v% solid-phase lipids, approximately 0.4 w / v% cationic lipids, approximately 0.5 w / v% sorbitan ester, and approximately 0.5 w / v% hydrophilic surfactant. Embodiment 65 A composition comprising a diluted or concentrated form of the composition described in any one of Embodiments 54 to 64. Embodiment 66 The composition according to Embodiment 65, wherein the composition according to any one of Embodiments 54 to 64 is diluted 2 to 30 times. Embodiment 67 The composition according to Embodiment 65, wherein the composition according to any one of Embodiments 54 to 64 is diluted 2 to 20 times. Embodiment 68 The composition according to Embodiment 65, wherein the composition according to any one of Embodiments 54 to 64 is diluted 2-fold. Embodiment 69 The composition according to Embodiment 65, wherein the composition according to any one of Embodiments 54 to 64 is concentrated 2 to 30 times. Embodiment 70 The composition according to Embodiment 65, wherein the composition according to any one of Embodiments 54 to 64 is concentrated 2 to 10 times. Embodiment 71 The composition according to any one of embodiments 54 to 70, wherein the liquid-phase lipid is a natural terpenoid or a synthetic terpenoid. Embodiment 72 The composition according to any one of Embodiments 54 to 70, wherein the liquid-phase lipid is natural squalene or synthetic squalene. Embodiment 73 The composition according to any one of Embodiments 54 to 72, wherein the solid phase lipid is a glycerolipid. Embodiment 74 The composition according to any one of Embodiments 54 to 72, wherein the solid phase lipid is a microcrystalline triglyceride. Embodiment 75 The composition according to any one of Embodiments 54 to 72, wherein the solid phase lipid is trimiristin. Embodiment 76 The composition according to any one of embodiments 54 to 75, wherein the cationic lipid is DOTAP. Embodiment 77 The composition according to any one of embodiments 54 to 76, wherein the sorbitan ester is sorbitan monostearate. Embodiment 78 The composition according to any one of embodiments 54 to 76, wherein the sorbitan ester is sorbitan monooleate. Embodiment 79 The composition according to any one of embodiments 54 to 76, wherein the sorbitan ester is sorbitan trioleate. Embodiment 80 The composition according to any one of embodiments 54 to 77, wherein the hydrophilic surfactant is polysorbate. Embodiment 81 The composition according to any one of embodiments 54 to 77, wherein the hydrophilic surfactant is polysorbate 80. Embodiment 82 The composition according to any one of Embodiments 1 to 20, Embodiments 34 to 41, Embodiments 45 to 51, and Embodiments 54 to 70, wherein the oily core comprises natural squalene or synthetic squalene and glycerolipids, the cationic lipid is DOTAP, the sorbitan ester is sorbitan monostearate or sorbitan monooleate, and the hydrophilic surfactant is polysorbate. Embodiment 83 The composition according to any one of Embodiments 1 to 20, Embodiments 34 to 41, Embodiments 45 to 51, and Embodiments 54 to 70, wherein the oily core comprises squalene and trimiristin, the cationic lipid is DOTAP, the sorbitan ester is sorbitan monostearate, and the hydrophilic surfactant is polysorbate 80. Embodiment 84 The composition according to Embodiment 83, wherein the NLC comprises about 2 w / v% to about 40 w / v% squalene, about 0.1 w / v% to about 10 w / v% trimyristine, about 0.2 w / v% to about 10 w / v% DOTAP, about 0.25 w / v% to about 5 w / v% sorbitan monostearate, and about 0.5 w / v% to about 10 w / v% polysorbate 80. Embodiment 85 The composition according to Embodiment 83, wherein the NLC comprises about 2 w / v% to about 6 w / v% squalene, about 0.1 w / v% to about 1 w / v% trimyristine, about 0.2 w / v% to about 1 w / v% DOTAP, about 0.25 w / v% to about 1 w / v% sorbitan monostearate, and about 0.5 w / v% to about 5 w / v% polysorbate 80. Embodiment 86 The composition according to Embodiment 85, wherein the NLC comprises about 3.75 w / v% squalene, about 0.25 w / v% trimyristine, about 3 w / v% DOTAP, about 3.7 w / v% sorbitan monostearate, and about 3.7 w / v% polysorbate 80. Embodiment 87 The composition according to any one of Embodiments 1 to 20, Embodiments 34 to 41, Embodiments 45 to 51, and Embodiments 54 to 70, wherein the oily core comprises natural squalene or synthetic squalene and glycerolipids, the cationic lipid is DOTAP, the sorbitan ester is sorbitan monostearate, sorbitan monooleate, or sorbitan trioleate, and the hydrophilic surfactant is polysorbate. Embodiment 88 The composition according to any one of Embodiments 1 to 20, Embodiments 34 to 41, Embodiments 45 to 51, and Embodiments 54 to 70, wherein the oily core comprises squalene and trimiristin, the cationic lipid is DOTAP, the sorbitan ester is sorbitan monostearate, sorbitan monooleate, or sorbitan trioleate, and the hydrophilic surfactant is polysorbate 80. Embodiment 89 The composition according to Embodiment 87 or Embodiment 88, wherein the NLC comprises about 2 w / v% to about 40 w / v% squalene, about 0.1 w / v% to about 10 w / v% trimyristine, about 0.2 w / v% to about 10 w / v% DOTAP, about 0.25 w / v% to about 5 w / v% sorbitan monostearate, sorbitan monooleate, or sorbitan trioleate, and about 0.2 w / v% to about 10 w / v% polysorbate 80. Embodiment 90 The composition according to Embodiment 87 or Embodiment 88, wherein the NLC comprises about 2 w / v% to about 6 w / v% squalene, about 0.1 w / v% to about 1 w / v% trimyristine, about 0.2 w / v% to about 1 w / v% DOTAP, about 0.25 w / v% to about 1 w / v% sorbitan monostearate, sorbitan monooleate, or sorbitan trioleate, and about 0.2 w / v% to about 5 w / v% polysorbate 80. Embodiment 91 The composition according to Embodiment 87 or Embodiment 88, wherein the NLC comprises about 4 w / v% squalene, about 0.25 w / v% trimyristine, about 0.4 w / v% DOTAP, about 0.5 w / v% sorbitan monostearate, sorbitan monooleate, or sorbitan trioleate, and about 0.5 w / v% polysorbate 80. Embodiment 92 A method for inducing an immune response in a subject, comprising administering a therapeutically effective amount of a composition described in any one of Embodiments 1 to 91 to a subject in need thereof, wherein the bioactive agent is a protein antigen or a nucleic acid molecule encoding a protein antigen. Embodiment 93 The method according to Embodiment 92, wherein the biological agent is RNA. Embodiment 94 The method according to Embodiment 92 or Embodiment 93, wherein the administration of the composition is intramuscular, parenteral, or intradermal. Embodiment 95 A method for inducing an immune response in a subject, comprising: (a) administering a therapeutically effective amount of an oncolytic virus encoding a protein antigen to a subject in need; and (b) administering a therapeutically effective amount of a composition described in any one of Embodiments 1 to 91 to the subject, wherein the bioactive agent is the protein antigen or a nucleic acid molecule encoding the protein antigen. Embodiment 96 The method according to Embodiment 95, wherein the administration of (a) and the administration of (b) are performed with an interval of at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 6 weeks, at least 2 months, at least 3 months, at least 6 months, or at least 1 year. Embodiment 97 A method for delivering a bioactive agent to cells, comprising contacting the cells with a composition described in any one of Embodiments 1 to 91. Embodiment 98 The method according to Embodiment 97, wherein the bioactive agent is a nucleic acid. Embodiment 99 The method according to Embodiment 97 or Embodiment 98, wherein contacting the cells with the composition involves administering the composition to a subject, the cells being present in the subject. Embodiment 100 A method for optimizing the delivery of a bioactive agent to cells, comprising selecting a molar ratio of the bioactive agent to NLC that optimizes the antibody titer induced in a subject including the cells. Embodiment 101 The method according to Embodiment 100, wherein the NLC is an NLC particle according to any one of Embodiments 1 to 91. Embodiment 102 A method for preparing the composition described in any one of Embodiments 1 to 91, (a) Mixing the solid phase lipid, the liquid phase lipid, the cationic lipid, and the hydrophobic surfactant to form an oil phase mixture; (b) Mixing the hydrophilic surfactant with water to form an aqueous phase mixture; (c) Mixing the oil phase mixture with the aqueous phase mixture to form the NLC particles; and (d) optionally, combining the bioactive agent with the NLC particles such that the bioactive agent binds to the surface of the NLC particles by non-covalent interactions or reversible covalent interactions. The method, including the method described above. Embodiment 103 The composition according to any one of Embodiments 1 to 91, wherein the biological agent is RNA, and the RNA encodes one or more TB antigens. Embodiment 104 The composition according to Embodiment 103, wherein the RNA encodes one or more TB antigens selected from Rv3619, Rv2389, Rv3478, and Rv1886. Embodiment 105 The composition according to Embodiment 104, wherein the RNA codes for the TB antigens Rv3619, Rv2389, Rv3478, and Rv1886. Embodiment 106 The method according to any one of embodiments 92 to 99, wherein the biological agent is RNA, and the RNA encodes one or more TB antigens. Embodiment 107 The method according to Embodiment 106, wherein the RNA encodes one or more TB antigens selected from Rv3619, Rv2389, Rv3478, and Rv1886. Embodiment 108 The method according to Embodiment 107, wherein the RNA codes for the TB antigens Rv3619, Rv2389, Rv3478, and Rv1886. Embodiment 109 The composition according to any one of Embodiments 1 to 91, wherein the bioactive agent is an adjuvant. Embodiment 110 The composition according to Embodiment 109, wherein the adjuvant is selected from TLR agonists, Rig-I agonists, saponins, sugars, sugar polymers, complex carbohydrates, whole virus particles, virus-like particles, virus fragments, and cell fragments. Embodiment 111 The composition according to Embodiment 110, wherein the adjuvant is selected from a TLR agonist and a Rig-I agonist. Embodiment 112 The composition according to Embodiment 111, wherein the TLR agonist is a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR7 agonist, a TLR8 agonist, or a TLR9 agonist. Embodiment 113 The composition according to Embodiment 111, wherein the TLR agonist is a TLR3 agonist. Embodiment 114 The composition according to any one of Embodiments 109 to 112, wherein the bioactive agent is selected from double-stranded RNA, RIBOXXOL, Poly(I:C), and Hiltonol®. Embodiment 115 A composition comprising an adjuvant in a stable emulsion, wherein the adjuvant is selected from a TLR3 agonist and a Rig-I agonist, and the stable emulsion is an oil-in-water emulsion. Embodiment 116 The composition according to Embodiment 115, wherein the emulsion contains 2-10% oil. Embodiment 117 The composition according to Embodiment 115 or Embodiment 116, wherein the oil is squalene. Embodiment 118 The composition according to any one of Embodiments 115 to 117, wherein the oil-in-water emulsion contains 0.01% to 0.1% α-tocopherol. Embodiment 119 The composition according to Embodiment 118, wherein the ratio of oil to α-tocopherol is greater than 1. Embodiment 120 The composition according to any one of Embodiments 115 to 118, wherein the oil-in-water emulsion contains 0.3 to 3% of a surfactant. Embodiment 121 The composition according to Embodiment 120, wherein the surfactant is polyoxyethylene sorbitan monooleate or poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene). Embodiment 122 The composition according to any one of Embodiments 115 to 121, wherein the oil-in-water emulsion contains about 1% Span85. Embodiment 123 The composition according to any one of Embodiments 115 to 121, wherein the oil-in-water emulsion contains 0.5% to 5%, 0.5% to 5%, 0.5% to 3%, or 1% to 3% of 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC). Embodiment 124 The composition according to any one of Embodiments 115 to 123, wherein the oil-in-water emulsion contains 0.5% to 5%, 0.5% to 5%, 0.5% to 3%, or 1% to 3% glycerol. Embodiment 125 A method for inducing or enhancing an immune response, comprising administering a therapeutically effective amount of a composition according to any one of embodiments 109 to 124 to a subject requiring the method. Embodiment 126 The method according to Embodiment 125, wherein the immune response is greater than the immune response when the subject is administered the adjuvant alone. Embodiment 127 The method according to Embodiment 125 or Embodiment 126, wherein the administration of the composition is intramuscular, parenteral, or intradermal. Embodiment 128 A composition comprising (a) an adjuvant and (b) an oil-in-water emulsion or nanoparticle lipid carrier (NLC) particles, wherein the oil-in-water emulsion or NLC particles contain squalene. Embodiment 129 The composition according to Embodiment 128, wherein the adjuvant is selected from TLR agonists, Rig-I agonists, saponins, sugars, glycopolymers, complex carbohydrates, whole virus particles, virus-like particles, virus fragments, and cell fragments. Embodiment 130 The composition according to Embodiment 129, wherein the adjuvant is selected from a TLR agonist and a Rig-I agonist. Embodiment 131 The composition according to Embodiment 130, wherein the TLR agonist is a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR7 agonist, a TLR8 agonist, or a TLR9 agonist. Embodiment 132 The composition according to Embodiment 131, wherein the TLR agonist is a TLR3 agonist. Embodiment 133 The composition according to any one of Embodiments 128 to 132, wherein the bioactive agent is selected from double-stranded RNA, RIBOXXOL, Poly(I:C), and Hiltonol®. Embodiment 134 A method for inducing or enhancing an immune response, comprising administering a therapeutically effective amount of a composition described in any one of embodiments 128 to 133 to a subject requiring the method. Embodiment 135 The method according to Embodiment 134, wherein the immune response is greater than the immune response when the subject is administered the adjuvant alone. Embodiment 136 The method according to Embodiment 134 or Embodiment 135, wherein the administration of the composition is intramuscular, parenteral, or intradermal.

[0407] The following examples are illustrative and not intended to be limiting. [Examples]

[0408] Example 1: Development of NLC formulation Nanostructured lipid carrier (NLC) compositions were prepared using combinations of emulsifiers, and the stability of the obtained compositions under storage conditions (5°C) was evaluated by particle size measurement. The oil phase consisted of squalene, and the liquid phase of the oily core consisted of a nonionic sorbitan ester surfactant, either sorbitan trioleate (Span® 85) or sorbitan monostearate (Span® 60), a cationic lipid DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride), and, in the case of NLC formulations, a solid lipid (glyceryl trimiristate-Dynasan® 114). The aqueous phase was a 10 mM sodium citrate trihydrate buffer containing the nonionic pegylated surfactant Tween® 80.

[0409] The composition of the formulations shown in each example is shown in Table 2 below. [Table 2]

[0410] To synthesize the NLC formulation, the oil phase was first prepared by mixing liquid-phase lipids, solid-phase lipids, positively charged lipids, and a hydrophobic surfactant in a Blend Vessel and then immersing it in an ultrasonic water bath (70±5°C) to promote solubilization. For the preparation of the aqueous phase, a hydrophilic surfactant, preferably Tween 80, was diluted with ultrapure water for injection (WFI) or a buffered aqueous solution (such as 10 mM sodium citrate trihydrate), and then stirred to completely dissolve it. This aqueous composition was heated to 60-70°C in an ultrasonic bath or the like before mixing it with the oil phase. In some cases, both phases were heated separately to 60°C in an ultrasonic bath. The oil phase and aqueous phase were mixed by highly shearing the multiphase mixture using a high-shear mixer. The mixing speed in a high-speed laboratory emulsifier (Silverson Machines, Inc.) was gradually increased to 5,000 RPM and then to a maximum of 10,000 RPM, after which mixing was performed for 10 minutes to 1 hour to obtain a crude mixture containing micron-sized oil droplets. The position of the Silverson mixing probe was adjusted as needed to ensure uniform oil dispersion and complete emulsification. The particle size was further reduced by high-shear homogenization in an M-110P microfluidizer (Microfluidics, Corp.). NLC particles were obtained from this crude emulsion using an M-110P Microfluidizer Materials Processor (Microfluidics). Each emulsion was treated in the microfluidizer by circulating it approximately five times under conditions of 45°C and 30,000 psi. The final pH was 6.5 to 6.8. To collect the final NLC formulation, the obtained NLC particle suspension was filtered using a 0.2 μm sterilization filter (e.g., a 0.2 μm polyethersulfone membrane syringe filter), stored at 2-8°C, and then the particle size was evaluated.

[0411] To evaluate the stability of NLC (uncompounded NLC), the mean hydrodynamic diameter (Z-mean) and polydispersity index (PDI) of each formulation were measured using dynamic light scattering (DLS) after storage at 5°C for various durations (Table 3). [Table 3]

[0412] Figures 1A to 1E show a comparison of the z-mean particle size of each formulation incubated at different temperatures, measured using dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments). The formulations were triplet diluted 1:100 with water and measured in disposable polystyrene cuvettes (SOP parameters: refractive index (RI) of the material = 1.59, RI of the dispersant (water) = 1.33, temperature (T) = 25°C, viscosity (water) = 0.887 cP, measurement angle = 173° backscattering, measurement position = 4.65 mm, auto-attenuation). For zeta potential measurement, the formulations were triplet diluted 1:100 and added to disposable DTS1070 (Malvern Instruments) folded capillary cells. The following SOP parameters were used: material RI = 1.59, dispersant RI (water) = 1.33, viscosity (water) = 0.887 cP, T = 25°C, auto-decay, and voltage selection. Table 3 lists the intensity-weighted Z-average particle size, PDI and zeta potential values ​​for each formulation, and the average from 3 measurements / replicate (total of 9 measurements). Particle size of formulated RNA (NLC+RNA complex or CNE+RNA complex) at different N:P values ​​was measured in triplicates in a 384-well plate using a Zetasizer Auto Plate Sampler (APS, Malvern Instruments). Zeta potential of formulated RNA at different N:P values ​​was measured in triplicates using the same method as above for each formulation individually. NLC binding ability was measured using a gel delay assay. Briefly, NLC and rvRNA complexes were prepared at various N:P values ​​and subjected to 1% agarose gel electrophoresis. Using standard concentrations, unbound or excess rvRNAs migrating through the gel were quantified.

[0413] Figure 16B compares the particle size distribution of NLC and CNE formulations during manufacturing, as measured by dynamic light scattering (DLS). Since long-term colloidal stability was a prerequisite for developing formulations suitable for storage in rapid response scenarios, the average particle size of formulations stored at 25°C was monitored. The particle size of NLC remained virtually unchanged for at least 9 months (less than 3% change), while CNE increased by 30% after 3 months of storage and by 350% after 9 months (Figure 16C). Nonionic surfactants, including hydrophobic sorbitan esters (Span), hydrophilic ethoxylated sorbitan esters (Tween), and cationic lipids (DOTAPs) are important for preserving colloidal stability, and because they are present at the interface, they play a crucial role in regulating biophysical interactions. Therefore, we attempted to experimentally elucidate the role of surfactants in rvRNA protection, protein expression, and immunogenicity mediation. NLCs with different physicochemical properties were synthesized using high-pressure microfluidization (see Methods). Table 2 summarizes the compositions of exemplary NLC and CNE formulations manufactured in-house according to a previously published method (see Brito et al. Mol. Ther. 22(12):2118-29 (2014)). Increasing the surfactant:oil (S:O) molar ratio increased the particle size (Figure 35), which allowed for the production of unimodal NLC with a Z-average particle size in the range of 40 nm to 100 nm when measured by DLS. The zeta potential correlated with the DOTAP content, increasing from approximately +15 mV with 0.4 w / v% DOTAP to +28 mV with 3.0 w / v% DOTAP.

[0414] The development of particle size as a function of time provides information about colloidal stability. Incubation at 5°C and 25°C simulates typical storage conditions (Figures 1A and 1B), 37°C simulates physiological temperature (Figure 1C), and 60°C and 80°C expose the formulations to high-temperature stress to promote colloidal stability and facilitate differentiation between formulations (Figures 1D and 1E). According to the latest data available at the time, the NLC formulation QG807 is stable at 60°C for at least one month. On the other hand, the particle size of the cationic nanoemulsion (CNE) QG386 more than tripled after incubation at 60°C for just 7 days. The particle size data at 60°C indicates that NLC significantly improved colloidal stability compared to CNE. See Figure 1D. Example 2: Evaluation of NLC particle size and oil:surfactant ratio

[0415] NLC consists of a hydrophobic core containing liquid oil and solid lipids, and a surfactant (also known as an emulsifier or emulsion agent) that forms an interface separating the hydrophobic phase (liquid oil and solid lipids, collectively referred to as oil in this specification) from the aqueous phase. Since the surfactant is present on the surface of the NLC nanoparticles, its content determines the total usable surface area. The oil, on the other hand, is present in the core and primarily contributes to the total usable volume. As a result, an increase in the surfactant / oil ratio increases the surface area (SA) / volume (V) ratio; that is, if the volume of the substance is fixed, an increase in the SA / V ratio means a decrease in the NLC particle size. The latter is experimentally shown in Figures 2A and 2B, where NLC produced under the same processing conditions, but with different oil / surfactant ratios, showed an increase in particle size as the oil / surfactant ratio increased. Particle size has a linear correlation with the oil / surfactant ratio (R 2 =0.97); or, particle size is inversely correlated with the surfactant / oil ratio (R 2 (=0.97). Example 3: Measurement of N / P ratio

[0416] The nitrogen:phosphate (N / P) ratio is theoretically expressed as the stoichiometric molar ratio of cationic nitrogen (positive charge) and anionic phosphate group (negative charge) available for the formation of the RNA-NLC complex. The cationic lipid DOTAP chloride (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride) used in NLC contains a quaternary trimethylammonium head group and has a positive charge regardless of pH. Since each DOTAP molecule has one trimethylammonium head group, the nitrogen concentration (or amount of positive charge) is essentially equal to the DOTAP concentration. On the other hand, each ribonucleotide monophosphate in an RNA copy consists of only one molecule, approximately proportional to the RNA concentration normalized to the average molecular weight of ribonucleotide monophosphates (339.5 g / mol). That is,

number

[0417] Furthermore, RNA bound to NLC was characterized as a function of theoretical N / P using a gel delay assay (GRA). 20 μg / mL RNA was mixed with either the undiluted or diluted formulation at a 1:1 (v / v) ratio. The dilution ratios ranged from 1 / 2 to 1 / 1600. Depending on the starting DOTAP concentration, the N / P ratio ranged from 0.12 (e.g., when 20 μg / mL RNA was complexed with 800-fold diluted QG807 (0.4 w / v% DOTAP)) to approximately 750 (when 20 μg / mL RNA was complexed with undiluted QG942 (3 w / v% DOTAP)). The RNA-NLC mixtures were complexed on ice for 30 minutes, followed by 1% agarose gel electrophoresis at 120 V for approximately 1 hour. Optical densitometry analysis of RNA bands was performed to determine the relative amount of RNA bound to NLC formulations as a function of N / P (Figure 3A). Except for QG807 and QG911, the percentage of RNA bound to NLC changed abruptly from 0% at N / P values ​​less than 1 to almost 100% at N / P values ​​greater than 1. At an N / P value of 1, where positive and negative charges are theoretically equimolar, approximately 50% of the RNA is bound to NLC. This suggests that the RNA-NLC binding reaction reaches an equilibrium state around an N / P value of 1, meaning that under these conditions, approximately equal amounts of bound and unbound RNA exist.

[0418] To understand the correlation between RNA-NLC binding, delivery, and expression, in vitro experiments were performed using SEAP expression replicons. SEAP replicons were complexed with either the QG807, QG843, QG942, or QG963 formulations. The N / P ratio of each formulation was varied, as shown in Figure 3A. Figures 3B–3E, particularly the SEAP data for QG942, QG963, and QG843, show that despite nearly identical RNA-NLC binding curves, the peak expression differs for each formulation.

[0419] Furthermore, in vitro experiments were conducted to determine NLC at various dilutions. v2The RNA / pharmaceutical complex was formed with 20 μg / mL SEAP rvRNA to obtain a series of N:P molar ratios. The physical and biological properties of these RNA / pharmaceutical complexes were determined by measuring in vitro SEAP expression, particle size, zeta potential, and RNA binding (Figures 22A-22D). The results indicate that there exists an N:P ratio that yields an optimal SEAP expression level corresponding to maximum RNA binding, a constant positive zeta potential, and minimal particle size increase. Considering the correlation between antigen expression and immunogenicity with the same formulation (see Pepini et al., J. Immunol. 1601877 (2017)), we hypothesized that similar neutralizing antibody titers can be expected at N:P ratios corresponding to peak SEAP expression (gray-shaded area in Figure 22).

[0420] From the perspectives of protein expression, reactogenicity, and immunogenicity, NLC is effective in vivo. v2 To characterize it, 5.8Log 10 We decided to test 4-5 N:P ratios, including those correlated with in vitro SEAP activity of RLU or higher (100, 37, 15, 5.6), and 3 N:P ratios outside the assumed optimal zone. To characterize protein expression, C57BL / 6 mice were intramuscularly injected with 1000 ng, 100 ng, and 10 ng doses of SEAP rvRNA for each N:P ratio. In addition, NLC was performed for each N:P ratio to characterize reactance. v2 50 μg of rvRNA complexed with was injected intradermally into guinea pigs, and the diameter of the redness was measured. 24 hours after injection, blood was collected from mice and serum SEAP activity was measured, and the injection site was measured in guinea pigs (Figures 22E-22H). To characterize immunogenicity, ZIKV rvRNA was subjected to NLC in each N:P ratio. v2 The substance was combined with another substance, and 1000 ng or 100 ng was delivered to mice via the intramuscular route. Blood was then collected from the mice 14 days later, and the neutralizing antibody titer was quantified (Figures 22F and 22G).

[0421] Starting with in vivo SEAP expression, the optimal N:P ratio was dose-dependent, with the optimal N:P being 15 at a 1000 ng dose, 37 at a 100 ng dose, and 100 at a 10 ng dose (Figure 22E). As expected, immunogenicity appeared to correlate with SEAP expression at the two doses compared (Figures 22F and 22G). At the 1000 ng dose, no significant difference in neutralizing antibody titers was detected at any of the N:P ratios tested, and this was similar to the SEAP activity at those N:P ratios (Figure 22F). At the 100 ng dose, a significant difference in neutralizing antibody titers was observed between N:P ratios 37 and 15, as well as a small but not significant difference in SEAP activity. However, a significant decrease in titer occurred between N:P ratios 15 and 5.6, similar to the decrease in SEAP activity (Figure 22G). Regarding reactogenicity, a significant reduction in redness diameter was observed when the N:P ratio was reduced from 37 to 15 (a 1 / 2.5-fold decrease) (Figure 22H). Importantly, these data suggest that while reactogenicity is significantly reduced even when the N:P ratio is reduced from 37 to 15 (a 1 / 2.5-fold decrease), the impact on antigen expression levels and subsequent immunogenicity is minimal, especially with high-dose rvRNA. In fact, there was no significant difference in SEAP activity between the 1000 ng and 100 ng doses at N:P values ​​of 15 and 37, indicating a dose-saving effect (Figure 22E). The largest difference in SEAP activity between doses was observed at low N:P ratios (Figure 22E).

[0422] To evaluate whether the physical state of RNA-formulation complexes can be predicted by the theoretical RNA / particle ratio, the hydrodynamic diameter (z-mean; nm) of three formulations, QG752, QG768, and QG386, mixed with a fixed amount (1 μg) of model 10 kbRNA in various amounts was compared. The hydrodynamic diameter for all formulations showed a profile close to a bell curve as the formulation dilution increased, i.e., N / P decreased (Figure 4A), RNA / particle increased (Figure 4B), or DOTAP / RNA decreased (Figure 4C). Despite all formulations having the same DOTAP content (0.4 w / v%), the particle size peak (potentially indicating clustering of RNA-crosslinked NLC particles) is observed at different N / P ratios (Figure 4A), namely, approximately 3 for QG768 and QG386 (40-fold formulation dilution or 0.01 w / v% DOTAP) and approximately 1 for QG752 (100-fold formulation dilution or 0.004 w / v% DOTAP). However, this difference is resolved by plotting the Z-mean as a function of RNA / particle ratio, taking into account the difference in starting particle size (Figure 4B), in which case the peak particle size is observed for all formulations when there are approximately 1-2 copies of RNA per NLC particle. Because QG752 has a starting average particle size (75 nm) smaller than both QG386 and QG768 (approximately 100 nm), it has a higher surface area / volume ratio, requiring more dilution (lower N / P) to achieve the same RNA / particle ratio as QG386 and QG768. Generally, when the chemical composition is kept constant, a decrease in particle size increases the surface area / volume ratio, resulting in an increase in particle concentration. This principle of increased particle concentration can be arithmetically summarized as follows: Formulations A and B share the same chemical composition and spherical shape, but have different diameters d A and diameter d B Each has, d A >d B If this is the case, formulation B is better than formulation A (d A / d B ) 3 It has twice the particle concentration.

[0423] Furthermore, this RNA formulation particle size data correlates with the RNA binding ability of the formulation measured by the Gel Delay Assay (GRA). The GRA is based on the principle that when the gel is separated under a certain applied voltage, the movement of unbound, or free, RNA matches that of the control, unformulated RNA, i.e., RNA alone. In other words, the GRA evaluates the binding and immobilization ability of the formulation to RNA under standard gel electrophoresis conditions. In the GRA, electrophoresis is performed in a one-piece precast 1.2% agarose gel stained with ethidium bromide (EtBr) (E-gel®, 1.2% general-purpose agarose, Thermo Fisher Scientific). When RNA was complexed with the NLC formulation QG768, immobilization was maintained at all N / P ratios higher than 2.3, which corresponds to the upward region (left to right) in Figure 4A, and this region contains the maximum value of the RNA formulation particle size profile for QG768. As the N / P ratio falls below 0.9, the amount of unbound RNA gradually increases, corresponding to the downward-sloping region (left to right) in Figure 2A. Therefore, particle size correlates with binding ability and can be used as a method to optimize RNA complex formation and delivery using the smallest possible formulation dose. To investigate how the RNA / particle ratio affects immunogenicity, mice (n=5 / group) were intramuscularly injected with ZIKV rvRNA complexed with QG768 at various ratios, and after 14 days, ZIKV neutralizing antibody (PRNT) was tested. 80 The titer was measured. Example 4: Evaluation of NLC formulation as a Zika vaccine candidate

[0424] Lead candidates of NLC formulations that demonstrated physical stability were combined with synthetic replicated viral RNA (rvRNA) derived from a strain of Venezuelan encephalitis virus (VEEV, TC83 strain) in which the VEEV structural gene was substituted with the ZIKV PrM-E cassette. The RNA replicon system was developed to facilitate a heterologous prime-boost strategy. The formulated rvRNA was administered intramuscularly using a conventional needle. Several NLC formulations enhanced a robust immune response in vivo. material and method cell culture

[0425] C6 / 36 cells derived from the mosquito Aedes albopictus (ATCC, Rockville, Maryland, USA) were maintained at 29°C and 5% CO2 in Dulbecco's Minimum Essential Culture Medium (DMEM) containing 10% (V / V) heat-inactivated fetal bovine serum (FBS), sodium pyruvate (1 mM), penicillin (100 U / mL), streptomycin (100 μg / mL), and 1% (v / v) tryptothenic acid phosphate broth (Sigma-America, St. Louis, Missouri). Vero cells, BHK-21 cells, and 293T cells (ATCC, Manassas, Virginia) were grown in DMEM containing 10% (V / V) heat-inactivated FBS, sodium pyruvate (1 mM), penicillin (100 U / mL), and streptomycin (100 μg / mL) at 37°C and 5% CO2. Mycoplasma contamination testing was also performed on all cell lines. Plasmid construct

[0426] Dr. Scott Weaver provided us with a plasmid encoding the 5' and 3' untranslated regions (UTRs) behind the SP6 promoter, the non-structural gene of the Venezuelan encephalitis virus (VEEV) strain TC-83 (Figure 21A), and a highly sensitive GFP under the control of the VEEV subgenome promoter, referred to as pSP6-VEE-Rep-GFP. We also designed a reporter rvRNA as a control encoding secreted human embryonic alkaline phosphatase (SEAP) (Figure 21B).

[0427] Next, using standard cloning techniques, the SP6 promoter was replaced with the T7 promoter, and this was named pT7-VEE-Rep-GFP. Fragments encoding codon-optimized versions of the prM and E genes derived from the French Polynesian Zika virus (ZIKV) strain H / PF / 2013 were synthesized and cloned into pUC57 (GenScript). Using the Q5 Mutation Kit (New England Biolabs), the Kozak sequence was inserted, followed by a variant Japanese encephalitis virus (JEV) signal sequence (ss), or upstream of an isomorphic ZIKV signal sequence (ss), using the following primers: JEVss-FWD (gctggcctccctggctgtggtcattgcctgcgctggagcaGCCGAGGTGACCAGGAGAGG; SEQ ID NO: 13) and JEVss-REV (cacatgattgatccgg cactcctcttgcccatggcggcggcGTGAGCTGGCGGCGGGTG; Sequence ID 14), or ZIKVss-FWD(ggaatcgtgggcctgctgctgaccacagcaatggcaGCCGAGGTGACCAGGAGAGG; Sequence ID 15), and ZIKVss-REV(cacggatgtgtctgctcctctccgcatggcggcggcGTGAGCTGGCGGCGGGTG; Sequence ID 16). Next, this complex fragment encoding JEVss or ZIKVss followed by the prM and E genes of ZIKV was PCR amplified using ZIKV-prM-E-FWD (AATGGACTACgacatagtcgccgccgccatg; SEQ ID NO: 17) and ZIKV-prM-E-REV (GCGGTTTTTGACAccgcggTCAGGCAGACACGGCG; SEQ ID NO: 18) as primers. It was then cloned between the PflFI and SacII sites of pT7-VEE-Rep-GFP using an In-Fusion enzyme mix (Clontech) to obtain pT7-VEE-Rep-JEVss-ZIKV-prM-E plasmid or pT7-VEE-Rep-ZIKVss-ZIKV-prM-E plasmid.pT7-VEE-Rep-SEAP was constructed by PCR amplification and cloned between the PflFI and SacII sites of pT7-VEE-Rep-GFP as described above (Figure 21B). All plasmids were confirmed by Sanger sequencing. RNA generation

[0428] Top10 cells (Invitrogen) were transformed and amplified, and then isolated using the Maxi-prep kit (Qiagen). Plasmids were linearized by restriction enzyme digestion with NotI enzyme (New England Biolabs) and purified with phenol-chloroform. RNA was transcribed in vitro using the T7 MEGAscript kit (Invitrogen), precipitated with lithium chloride, and capped using the Vaccinia Capping kit (New England Biolabs). The capped transcript was then precipitated with lithium chloride, resuspended in nuclease-free water to a final concentration of 1 μg / μL, and analyzed by agarose gel electrophoresis. All RNA was aliquoted and stored at -80°C. Ribonuclease Challenge Assay

[0429] ZIKV-rvRNA is NLC v1 and NLC v2 Then, composites were formed so that the N:P ratios were 50 and 15, respectively, and left on ice for 30 minutes. NLC v2After diluting the complex with nuclease-free water, the complex containing 1 μg of rvRNA at a concentration of 20 μg / mL was treated with 50 ng of RNase A (Thermo Scientific) for 30 minutes at room temperature, followed by incubation with 5 μg of recombinant proteinase K (Thermo Scientific) at 55°C for 10 minutes. RNA was then extracted using equal volumes of phenol:chloroform:isoamyl alcohol (25:24:1) (Invitrogen). After vortexing, the sample was centrifuged at 17,000 × g for 15 minutes. The supernatant was collected, mixed 1:1 with glyoxal load dye (Invitrogen), and heated at 50°C for 15 minutes. RNA equivalent to 200 ng was added and electrophoresis was performed on a denatured 150 mL 1% agarose gel in Northern Max Gly electrophoresis buffer (Invitrogen) at 120 V for 45 minutes. The gel was imaged using the ChemiDoc™ MP imaging system (Bio-Rad). The intensity of intact rvRNA bands was compared to phenol:chloroform:isoamyl extracted RNA from a complex that had not been treated with ribonuclease and protease K. Additional controls included rvRNA treated with ribonuclease and protease K alone, and rvRNA that had not been treated, both with an added 200 ng of rvRNA. Complexation conditions for in vitro and in vivo experiments

[0430] In N:P optimization experiments, NLC v1 or NLC v2The rvRNA was serially diluted with 10 mM citrate buffer and complexed 1:1 with rvRNA diluted to 20 μg / mL in nuclease-free 10% sucrose solution (to maintain isotonicity without using ionic agents such as physiological saline). The rvRNA was added to the formulation and thoroughly mixed by gentle pipetting. This complex was incubated on ice for 30 minutes to obtain a series of N:P molar ratios. These complexes were then further diluted with 10% sucrose to obtain the desired dose. For in vitro human PBMC stimulation, the complexed formu...

Claims

1. A composition comprising nanostructured lipid carrier (NLC) particles for delivering a bioactive agent to cells, (a) an oily core containing a mixture of liquid-phase lipids and solid-phase lipids, (b) Cationic components including cationic lipids, (c) Hydrophobic surfactant containing sorbitan ester, (d) A hydrophilic surfactant having a molar ratio of 0.2 to 1.5 with respect to the cationic component, and (e) Bioactive agents Includes, The liquid-phase lipid is squalane, the solid-phase lipid is glyceryl trimyristate, the cationic component is DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride), the hydrophobic surfactant is sorbitan trioleate or sorbitan monostearate, the hydrophilic surfactant is polysorbate, and the biological agent is RNA. The aforementioned composition.

2. The composition according to claim 1, wherein the bioactive agent is bound to the NLC particles.

3. The composition according to claim 1 or claim 2, which delivers the bioactive agent to the cells.

4. The composition according to any one of claims 1 to 3, wherein the hydrophobic surfactant is present in an amount sufficient to enhance the ability of the composition to deliver the bioactive agent to the cells when compared with a control composition that does not contain the surfactant.

5. The composition according to any one of claims 1 to 4, wherein the bioactive agent codes for a protein, or the bioactive agent codes for a protein antigen.

6. The composition according to claim 5, which, when administered to a subject in an effective amount, induces an immune response to the antigen that is equivalent to or greater than the immune response induced when the bioactive agent is administered to the subject without the NLC.

7. The composition according to any one of claims 1 to 6, wherein the polysorbate is polysorbate 80.

8. The composition according to any one of claims 1 to 7, wherein the mean polydispersity index of the NLC particles is 0.1 to about 0.

5.

9. The composition according to any one of claims 1 to 8, wherein the z-average particle size of the NLC particles is approximately 20 nm to approximately 200 nm, approximately 20 nm to approximately 150 nm, approximately 20 nm to approximately 100 nm, approximately 20 nm to approximately 80 nm, approximately 20 nm to approximately 60 nm, approximately 40 nm to approximately 200 nm, approximately 40 nm to approximately 150 nm, approximately 40 nm to approximately 100 nm, approximately 40 nm to approximately 80 nm, or approximately 40 nm to approximately 60 nm.

10. The composition according to any one of claims 1 to 9, wherein the z-average particle size of the NLC particles is about 40 nm to about 80 nm.

11. The composition according to any one of claims 1 to 10, wherein the oil-surfactant molar ratio is about 0.05 to about 12.

12. Hydrophilic surfactant: The composition according to any one of claims 1 to 11, wherein the ratio of cationic components is about 0.5 to about 1.

13. A composition according to any one of claims 1 to 12, comprising approximately 0.2 w / v% to approximately 40 w / v% liquid-phase lipids, approximately 0.1 w / v% to approximately 10 w / v% solid-phase lipids, approximately 0.2 w / v% to approximately 10 w / v% cationic lipids, approximately 0.25 w / v% to approximately 5 w / v% sorbitan monoester, and approximately 0.5 w / v% to approximately 10 w / v% hydrophilic surfactant.

14. An immune response generating agent comprising the composition according to any one of claims 1 to 13, wherein the bioactive agent encodes a protein antigen.

15. A method for preparing the composition according to any one of claims 1 to 14, (a) Mixing the solid phase lipid, the liquid phase lipid, the cationic lipid, and the hydrophobic surfactant to form an oil phase mixture; (b) Mixing the hydrophilic surfactant with water to form an aqueous phase mixture; and (c) Mixing the oil phase mixture with the aqueous phase mixture to form the NLC particles; The method, including the method described above.

16. The composition according to any one of claims 1 to 13, wherein the RNA encodes one or more TB antigens.

17. The composition according to any one of claims 1 to 13, further comprising an adjuvant.

18. The composition according to claim 17, wherein the adjuvant is selected from a TLR agonist, a Rig-I agonist, a saponin, a sugar, a sugar polymer, a complex carbohydrate, whole virus particles, virus-like particles, virus fragments, and cell fragments.

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