POLYMERIC COMPOSITION

Abstract

The invention relates to polymeric compositions, nanoparticles and vaccines comprising polymeric compositions. The invention extends to medical uses of the polymeric compositions, nanoparticles and vaccines. The invention further extends to methods of producing the polymeric compositions and nanoparticles.

Claims

1. A polymeric composition comprising a plurality of polymers of formula (I): ##STR00007## wherein L.sup.1 to L.sup.5 are each independently an optionally substituted C.sub.1-12 alkylene, an optionally substituted C.sub.2-12 alkenylene, an optionally substituted C.sub.2-12 alkynylene, an optionally substituted C.sub.3-6 cycloalkylene, an optionally substituted 3 to 8 membered heterocyclylene, an optionally substituted C.sub.6-12 arylene, an optionally substituted 5 to 10 membered heteroarylene or L.sup.6L.sup.7, wherein adjacent carbon atoms in the alkylene, alkenylene or alkynylene are optionally interrupted by one or more heteroatoms; L.sup.6 and L.sup.7 are independently an optionally substituted C.sub.1-12 alkylene, an optionally substituted C.sub.2-12 alkenylene, an optionally substituted C.sub.2-12 alkynylene, an optionally substituted C.sub.3-6 cycloalkylene, an optionally substituted 3 to 8 membered heterocyclylene, an optionally substituted C.sub.6-12 arylene or an optionally substituted 5 to 10 membered heteroarylene, wherein adjacent carbon atoms in the alkylene, alkenylene or alkynylene are optionally interrupted by one or more heteroatoms; R.sup.1 and R.sup.2 are each independently H, an optionally substituted C.sub.1-12 alkyl, an optionally substituted C.sub.2-12 alkenyl or an optionally substituted C.sub.2-12 alkynyl; R.sup.3 is OR.sup.4, COOR.sup.4, SO.sub.2OR.sup.4, (OCH.sub.2CH.sub.2).sub.mOH, or NR.sup.4R.sup.5, R.sup.4 and R.sup.5 are each independently H, an optionally substituted C.sub.1-12 alkyl, an optionally substituted C.sub.2-12 alkenyl, an optionally substituted C.sub.2-12 alkynyl, an optionally substituted C.sub.3-6 cycloalkyl, an optionally substituted 3 to 8 membered heterocyclyl, an optionally substituted C.sub.6-12 aryl or an optionally substituted 5 to 10 membered heteroaryl, wherein adjacent carbon atoms in the alkyl, alkenyl or alkenyl are optionally interrupted by one or more heteroatoms; and m is an integer between 1 and 10; or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof; characterised in that the average molecular mass of the plurality of polymers of formula (I) is greater than 5 kg mol.sup.?1.

2. The polymeric composition according to claim 1, wherein L.sup.1 to L.sup.4 are each independently an optionally substituted C.sub.1-6 alkylene, an optionally substituted C.sub.2-6 alkenylene or an optionally substituted C.sub.2-6 alkynylene.

3-14. (canceled)

15. A polymeric composition comprising self-amplifying RNA (saRNA) and a plurality of polymers of formula (I): ##STR00008## wherein L.sup.1 to L.sup.5 are each independently an optionally substituted C.sub.1-12 alkylene, an optionally substituted C.sub.2-12 alkenylene, an optionally substituted C.sub.2-12 alkynylene, an optionally substituted C.sub.3-6 cycloalkylene, an optionally substituted 3 to 8 membered heterocyclylene, an optionally substituted C.sub.6-12 arylene, an optionally substituted 5 to 10 membered heteroarylene or L.sup.6L.sup.7, wherein adjacent carbon atoms in the alkylene, alkenylene or alkynylene are optionally interrupted by one or more heteroatoms; L.sup.6 and L.sup.7 are independently an optionally substituted C.sub.1-12 alkylene, an optionally substituted C.sub.2-12 alkenylene, an optionally substituted C.sub.2-12 alkynylene, an optionally substituted C.sub.3-6 cycloalkylene, an optionally substituted 3 to 8 membered heterocyclylene, an optionally substituted C.sub.6-12 arylene or an optionally substituted 5 to 10 membered heteroarylene, wherein adjacent carbon atoms in the alkylene, alkenylene or alkynylene are optionally interrupted by one or more heteroatoms; R.sup.1 and R.sup.2 are each independently H, an optionally substituted C.sub.1-12 alkyl, an optionally substituted C.sub.2-12 alkenyl or an optionally substituted C.sub.2-12 alkynyl; R.sup.3 is OR.sup.4, COOR.sup.4, SO.sub.2OR.sup.4, (OCH.sub.2CH.sub.2).sub.mOH, or NR.sup.4R.sup.5, R.sup.4 and R.sup.5 are each independently H, an optionally substituted C.sub.1-12 alkyl, an optionally substituted C.sub.2-12 alkenyl, an optionally substituted C.sub.2-12 alkynyl, an optionally substituted C.sub.3-6 cycloalkyl, an optionally substituted 3 to 8 membered heterocyclyl, an optionally substituted C.sub.6-12 aryl or an optionally substituted 5 to 10 membered heteroaryl, wherein adjacent carbon atoms in the alkyl, alkenyl or alkenyl are optionally interrupted by one or more heteroatoms; and m is an integer between 1 and 10; or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof.

16-27. (canceled)

28. A method of producing a high molar mass poly(amido amine), the method comprising contacting a compound of formula (II): ##STR00009## with a compound of formula (III):
NHR.sup.12R.sup.13(III) in the presence of a Lewis base to thereby cause the compounds of formula (II) and formula (III) to undergo a polymerisation reaction and produce the high molar mass poly(amido amine); wherein R.sup.1 and R.sup.2 are each independently H, an optionally substituted C.sub.1-12 alkyl, an optionally substituted C.sub.2-12 alkenyl or an optionally substituted C.sub.2-12 alkynyl; R.sup.6 to R.sup.11 are each independently H, a halogen, an optionally substituted C.sub.1-12 alkyl, an optionally substituted C.sub.2-12 alkenyl, an optionally substituted C.sub.2-12 alkynyl, an optionally substituted C.sub.3-6 cycloalkyl, an optionally substituted 3 to 8 membered heterocyclyl, an optionally substituted C.sub.6-12 aryl or an optionally substituted 5 to 10 membered heteroaryl; L.sup.2 and L.sup.3 are each independently absent or an optionally substituted C.sub.1-12 alkylene, an optionally substituted C.sub.2-12 alkenylene, an optionally substituted C.sub.2-12 alkynylene, an optionally substituted C.sub.3-6 cycloalkylene, an optionally substituted 3 to 8 membered heterocyclylene, an optionally substituted C.sub.6-12 arylene, an optionally substituted 5 to 10 membered heteroarylene or L.sup.6L.sup.7, wherein adjacent carbon atoms in the alkylene, alkenylene or alkynylene are optionally interrupted by one or more heteroatoms; L.sup.8 is absent or is SS; R.sup.12 is an optionally substituted C.sub.1-12 alkyl, an optionally substituted C.sub.2-12 alkenyl, an optionally substituted C.sub.2-12 alkynyl, an optionally substituted C.sub.3-6 cycloalkyl, an optionally substituted 3 to 8 membered heterocyclyl, an optionally substituted C.sub.6-12 aryl, an optionally substituted 5 to 10 membered heteroaryl, wherein adjacent carbon atoms in the alkyl, alkenyl or alkenyl are optionally interrupted by one or more heteroatoms; and R.sup.13 is H, an optionally substituted C.sub.1-12 alkyl, an optionally substituted C.sub.2-12 alkenyl, an optionally substituted C.sub.2-12 alkynyl, an optionally substituted C.sub.3-6 cycloalkyl, an optionally substituted 3 to 8 membered heterocyclyl, an optionally substituted C.sub.6-12 aryl, an optionally substituted 5 to 10 membered heteroaryl, wherein adjacent carbon atoms in the alkyl, alkenyl or alkenyl are optionally interrupted by one or more heteroatoms.

29. The method of claim 28, wherein the compound of formula (II) is a compound of formula (IIa): ##STR00010##

30-32. (canceled)

Description

[0223] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:

[0224] FIG. 1 is a schematic illustration of (a) improved Aza-Michael addition to afford high molar mass poly(amido amine)s, poly(CBA-4-amino-1-butanol)s pABOLs with molar masses up to 167 kg mol.sup.1; (b) complexation with self-amplifying RNA (saRNA) via titration method and transfection efficacy of the pABOL-100 polyplexes, comparing to jetPEI and PEI MAX;

[0225] FIG. 2 shows the synthesis of high MW pABOL and characterization of resulting saRNA polyplexes. (a) Polymerization kinetics of ABOL with CBA under different reaction conditions. The conversion values were calculated from the integrals of doubled bond signals at 5.60?6.23 ppm, using the methylene signals at 1.36?1.47 ppm as the internal reference; (b) and (c) particle diameter and zeta potential of polyplexes form via the direct mixing method between pABOLs and saRNA at polymer to saRNA weight ratio ranging from 1 to 45. PEI-44 [poly(ethylene imine), linear, 44 kg mol.sup.?1] at weight ratio of 1 was used as the reference; and (d) typical TEM of polyplexes (pABOL-100/saRNA=45:1, w/w) stained with 2 wt % uranyl acetate (scale bar: 100 nm);

[0226] FIG. 3 shows the in vitro transfection efficiency and cytotoxicity of pABOL polyplexes. (a) Quantification of fLuc expression in relative light units (RLU) of polyplexes formed by PEI-44 and pABOLs with saRNA, 24 h after transfection at mass ratios ranging from 1:1 to 45:1 (w/w); (b) quantification of fLuc expression in RLU of polyplexes formed by all pABOLs in Table 1 at a mass ratio of 45:1 (see Figure S13 for other mass ratios); (c) cytotoxicity studies of polyplexes formed at mass ratios ranging from 10:1 to 450:1 (saRNA loading=100 ng), 24 h after initial transfection; and (d) quantification of fLuc expression in RLU of polyplexes, using untreated cells (?) and cells (+) treated with Glutathione (GSH) inhibitor, Buthionine sulphoximine (BSO);

[0227] FIG. 4 shows functional characterization of polyplexes prepared using either the direct addition or titration methods in vitro and in vivo. (a) Hydrodynamic diameter, polydispersity and zeta potential of polyplexes form via the titration method (adding saRNA to polymer) at various titration flow rates (from 10 to 160 ?L min.sup.?1), using pABOL-100 (polymer/RNA=45:1, w/w, see SI for details). Data from direct mixing method is included for reference; (b) absorbance curves of polyplexes measured by Nanodrop before (black) and after (red) passing through a 0.2 m syringe filter. (pink curve: absorbance of pABOL-100 in buffer with the same concentration as in the obtained polyplex solution); (c) hydrodynamic diameter of polyplexes measured by DLS before (black) and after (red) passing through a 0.2 ?m syringe filter; (d) and (e) quantification of fLuc expression in RLU, using filtered (+) and non-filtered (?) polyplexes formed via the direct mixing and titration method, respectively; and (f) quantification of fLuc expression of filtered or non-filtered pABOL polyplexes formed by direct addition and titration methods, 7 d after injection. Mice were injected with 5 ?g of saRNA in each leg, and a ratio polymer to RNA of 45:1 (w/w) for pABOL. Each circle represents one leg of one animal, and bar represents mean+/?SEM.; (g) Representative images of each group, corresponding to (f);

[0228] FIG. 5 shows the effect of molecular weight, route of administration and ratio of pABOL to RNA on in vivo expression of fLuciferase-encoding saRNA polyplexes. a-b) Quantification of fLuc expression of PEI (jetPEI and PEI MAX) and pABOL polyplexes in total flux (p/s), 7 d after injection. Mice were injected with 5 ?g of saRNA either (a) intramusculary or (b) intradermally, and a ratio polymer to RNA of 45:1 (w/w) for pABOL, 1:1 for PEI MAX, and an N:P of 8 for jetPEI. Each circle represents one leg of one animal, and bar represents mean+/?SD. c-d) Representative images of each group. e) Quantification of fLuc expression in vivo 7 d after IM injection of 5 ?g of saRNA with varying ratios of pABOL to RNA. Each circle represents one leg of one animal, and bar represents mean+/?SD. f) Representative images of each group, corresponding to e). * Indicates significance based on a one-way ANOVA with ?=0.05;

[0229] FIG. 6 shows cellular expression of saRNA after IM (mouse) or ID (human, mouse) injection with polyplex formulations. a) Percentage of eGFP+ cells out of total live cells for each formulation after an intradermal injection of 2 ?g of saRNA in human skin explants. Explants were analyzed 72 h after initial injection. jetPEI and PEI MAX were formulated at ratios of N/P=8 and 1, respectively. pABOL formulations were prepared at ratios of 10:1, 25:1, and 45:1. Bars represent mean+/?SD for n=3, with *, ** and *** indicating significance of p<0.05, 0.01 and 0.001, respectively; b-c) Percentage of eGFP+ cells out of total live cells for each formulation after either IM (b) or ID (c) injection of 5 ?g of saRNA in mice. Tissue was excised 7 d after initial injection. jetPEI was formulated at a ratio of N/P=8, and pABOL formulations were prepared at a ratio of 45:1. Bars mean+/?SD for n=8 (IM) and 4 (ID), with * indicating significance of p<0.05. d-f) Histograms of mean eGFP fluorescence intensity (MFI) for each formulation in human skin explants (d), IM injection in mice (e) and ID injection in mice (f). g) fLuc expression of human skin explants after ID injection with 2 ?g of saRNA. Explants were analyzed 72 h after initial injection. Bars represent mean+/?SD for n=3. h) Representative images corresponding to g);

[0230] FIG. 7 shows phenotypic identity of cell present in human skin explants and GFP+ cells after intradermal (ID) injection of polyplex formulations as determined by flow cytometry. a) Identity of cells in the population of total cells extracted from human skin explants; and b) identity of GFP-expressing skin cells from explants treated with polyplex formulated eGFP-encoding saRNA. Cells identified using the following antibodies: epithelial cells (CD45?), fibroblasts (CD90+), NK cells (CD56+), leukocytes (CD45+), Langerhans cells (CD1a+), monocytes (CD14+), dendritic cells (CD11c+), T cells (CD3+) and B cells (CD19+);

[0231] FIG. 8 shows immunogenicity of HA-encoding saRNA polyplexes. a-b) Change in body weight after IN challenge with Cal/09 flu virus for mice injected either IM (a) or ID (b). Dots represent mean percentage, normalized to Day 0 for each mouse, +/?SD for n=5; c-d) HA antigen-specific IgG antibody titers following immunization with prime and boost of saRNA complexed with jetPEI, 8 kg mol.sup.?1 pABOL or 100 kg mol.sup.?1 pABOL for mice injected either IM (c) or ID (d). Each bar represents mean+/?SEM for n=5 at each time point; and

[0232] FIG. 9 shows graphs monitoring the (a) molecular weight and (b) molecular weight dispersity (?) over time using SEC, corresponding to 1 M (?), 5 M (?) and 5 M with TEA (?).

EXAMPLES

[0233] Poly(amido amine)s (pAAs) fit the inventors' polymeric criteria and in addition, depending on the monomer combinations, linear pAAs generally have good water solubility, stability against hydrolysis and tunable degradation..sup.16 The use of a disulphide monomer, N,N-cystaminebisacrylamide (CBA), enables bioreduction via a disulphide backbone, which undergoes rapid cleavage intracellularly due to the presence of glutathione (GSH)..sup.16 Furthermore, preparation of pAAs is simple; two monomers are mixed together and undergo Aza-Michael polyaddition, which is a facile approach for scale-up and clinical translation. However, previous reports on pAAs reports polymers with molar mass limited to ?5 kg mol.sup.?1 with ?10 repeat units,.sup.16-19 which to be more accurate, are just oligomers.

[0234] As explained below, the inventors prepared a library of poly(CBA-4-amino-1-butanol) (pABOL) (see FIG. 1) with varying molar mass, ranging from 5 to 167 kg mol.sup.?1, using an optimized Aza-Michael polyaddition synthesis protocol. Using commercially available pEIs that have been used extensively in vitro and in vivo as a positive control,.sup.20-24 the inventors characterized the in vitro transfection efficiency and cytotoxicity. The inventors then devised a method of polyplex preparation that enables monodisperse particles and sterile filtration, which is imperative for clinical translation of this formulation. Furthermore, the inventors characterized the relationship between pABOL molar mass and protein expression in vivo using both intramuscular (IM) and intradermal (ID) injection. They then assessed whether protein expression was due to the quality or quantity of cellular expression ex vivo in human skin explants and in vivo in murine skin and muscle and phenotyped the cells in human skin that express pABOL/saRNA complexes. Finally, the inventors use pABOL and hemagglutinin (HA)-encoding saRNA as a vaccine model and observe the immunogenicity and ability to protect against influenza challenge compared to pEI in vivo.

Example 1Synthesis of pABOLs with High Molar Masses

[0235] First, the inventors increased the initial monomer concentration from 1.0 M to 5.0 M (defined as the CBA concentration). This led to a significant increase in reaction rate, reaching 98% of double bond conversion after 2 days with a M.sub.w of 8.7 kg mol.sup.?1 (FIG. 9a) in comparison with 4.9 kg mol.sup.?1 (conv. =94%) observed at 1.0 M. However, due to the high viscosity the reaction hit a kinetic barrier.

[0236] To address this issue, triethylamine (TEA) was employed as a Lewis base catalyst to further increase the reaction rate. The addition of TEA increased the conversion by 0.2% in 4 days (FIG. 1) and doubled the molecular mass compared to the non-catalysed reaction (FIG. 9a). With the combination of higher monomer concentration and use of TEA as a catalyst, the targeted conversions (>99.5%) can be easily achieved within 3 days. The conversions were not monitored after 4 days as the double bond conversion exceeded 99.9% in the catalysed reaction; thus the residual signals were too weak to be detected in the NMR spectroscopy even with 1024 scans. However, higher molar masses are accessible by extending reaction period from 5 to 14 days. pABOLs with molar masses ranging from 5 to 167 kg mol.sup.?1 (Table 1) were successfully prepared via the improved aza-Michael polyaddition conditions.

[0237] Thus, for the first time, the inventors were able to synthesize pABOLs with molar masses >30 kg mol.sup.?1. Moreover, the method described here may enable synthesis of high molar mass poly(amido amine)s given the broad range of commercial chemicals that undergo aza-Michael polyaddition.

TABLE-US-00001 TABLE 1 pABOLs with variable molar masses. # Polymers.sup.a M.sub.w (kDa).sup.b ?.sup.b 0 pABOL (ref.).sup.16, 25 ca. 5~6 ca. 1.1~1.4 1 pABOL-5.sup.c 5 1.7 2 pABOL-8 8 2.0 3 pABOL-18 18 2.5 4 pABOL-25 25 2.9 5 pABOL-33 33 4.6 6 pABOL-41 41 3.9 7 pABOL-72 72 5.9 8 pABOL-92 92 5.0 9 pABOL-100 105 6.4 10 pABOL-167 167 4.7 .sup.aPolymerization conditions: [CBA]/[ABOL]/[TEA] = 1.01/1/0.1, 5.0M in MeOH/H.sub.2O (4/1, v/v) at 45? C., for 5~14 d under N.sub.2 and in dark; .sup.bDetermined by SEC, in DMF, at 30? C., calibrated using poly(methyl methacrylate) standards with narrow ?; .sup.cPolymerized without TEA as the catalyst.

Example 2Increasing pABOL Molar Mass Enhances Transfection Efficiency of Nucleic Acids In Vitro

[0238] In order to assess the effect of polymer molar mass on complexation, the polyplexes were prepared via a direct mixing procedure. Given that the binding sites on both the saRNA and high molar mass pABOLs might not be completely accessible, due to the higher-order structure and the sterically hindered tertiary amine groups, respectively, the inventors opted to use a range of polymer/RNA weight ratios (from 1:1 to 60:1) instead of the commonly used N/P values. It is noteworthy that theoretical average molar masses per charge of pABOLs and saRNA are 349.5 g mol.sup.?1 and 339.5 g mol.sup.?1, respectively, suggesting the weight ratios are close to N/P values. When combined, pABOL and saRNA form nanoparticles with diameter ranging from 100 to 400 nm regardless of the weight ratios (FIG. 2b). However, only at weight ratios higher than 45:1 can nanoparticles exhibit sufficient positive surface charge to maintain sufficient colloidal stability as well as good cell permeability (FIG. 2c)..sup.26 The inventors observed that a polymer/RNA weight ratio of at least 10:1 (N/P=8) was required to reach a neutral surface charge, which confirms their assumption that due to the higher-order structure and steric hindrance a certain amount of binding sites are left unbound during the polyplex formation. Furthermore, it takes less polymer loading to reach a positive surface charge for pABOLs with higher molar mass. This is because polycations with higher molar mass have more effective binding sites per chain, leading to the increase in binding constant between the polymer chains and saRNA.sup.27 and consequently more polymer incorporated into nanoparticles and increased surface charge. This indicates that higher molar mass is favourable for reaching the desired surface charge with a lower polymer loading, which could also be beneficial for endosomal escape, as more tertiary amines per nanoparticles can serve as a proton sponge..sup.28 Transmission electron microscopy (TEM) was used to confirm the nanoparticle structure. The particle size (pABOL-100/saRNA=45:1, w/w) shown in FIG. 2d is smaller than the hydrodynamic diameter demonstrated in FIG. 2b, which is commonly observed and caused by dehydration during sample characterization.

[0239] In order to evaluate the effect of pABOL molar mass on the intracellular delivery of saRNA, the inventors used saRNA encoding firefly luciferase (fLuc) as a reporter protein and indicator of transfection efficiency (FIG. 3a). PEI is a polycation that widely used for nucleic acid transfection, and thus serves as a positive control..sup.29 At weight ratios below 10:1, there was no effect of increasing the molar mass, likely because of surface charge being negative or neutral, as shown in FIG. 1c, which is unfavourable for cell uptake or colloidal stability. However, at weight ratios >45:1, the transfection efficiency shows a molar mass dependencehigher molar masses enhance higher transfection efficacy. To investigate further, additional in vitro tests were conducted using pABOLs with a wider range of molar masses at weight ratio of 45:1 (FIG. 3b). The results confirm the molar mass dependence, but also suggest non-monotonic behavior. At molar masses below 72 kg mol.sup.?1, higher molar mass is indeed preferable. However, the transfection efficiency reaches a plateau for pABOLs with molar mass between 72 and 167 kg mol.sup.?1. Considering the added time it takes to increase the molar mass from 100 to 167 kg mol.sup.?1 during polymerization and negligible impact on transfection efficacy, there is no advantage in increasing the molar mass beyond 100 kg mol.sup.?1.

[0240] The inventors also tested whether increasing the molar mass of pABOL similarly enhanced the transfection efficiency mRNA and plasmid DNA (pDNA). Although the enhancement in mRNA and pDNA transfection was not as significant as in saRNA, it implies the molar mass effect is not only specifically applied to long-chain nucleic acids, like saRNA, but to other nucleic acid species as well. This knowledge is useful for the future design of polymer-based delivery systems for nucleic acids.

[0241] In addition to transfection efficiency, the inventors also evaluated the in vitro cytotoxicity of saRNA/pABOL formulations (FIG. 3c). Compared to PEI, pABOLs display less cytotoxicity. Furthermore, a molar mass dependence was observed for pABOLs. pABOLs with low/moderate molar masses (8 and 25 kg mol.sup.?1) demonstrate much less cytotoxicity, comparing to their high molar mass analogues (72 and 100 kg mol.sup.?1), which could be due to surface charge and/or the concentration of free polycations.

[0242] The inventors then sought to determine the role of bioreduction of pABOLs on in vitro transfection efficiency. As a bioreducible polycation, it is hypothesized that pABOL releases saRNA via the intracellular glutathione (GSH) reduction of the disulfide bonds on its backbone..sup.16 To confirm that pABOL is capable of being reduced by GSH, the bioreduction of pABOLs was monitored using GSH and the reduced product was identified to be a dithiol compound. The inventors then used a known GSH inhibitor, buthionine sulphoximine (BSO), 3? to pretreat cells and evaluate whether pABOLs had the same transfection with normal or reduced intracellular levels of GSH. All pABOL polyplexes showed a significant decrease in transfection efficiency following BSO pretreatment (FIG. 2d). PEI, which is not bioreducible, showed no decrease in transfection efficiency indicating that BSO pretreatment did not impact the ability of the cells to express luciferase, and that GSH is integral in decomplexation of pABOL.

[0243] This agrees with previous reports that the bioreducibility of pABOL accelerates the decomplexation with other nucleic acids..sup.18,31 This may be particularly relevant for self-amplifying RNAs, where the fast release mechanism delivered by pABOLs may facilitate rapid transgene expression.

Example 3Optimization of pABOL/saRNA Complexation Procedure for Sterile Filtration and Scale-Up

[0244] In order to facilitate downstream product sterilization, it is imperative to develop saRNA-polyplexes that can undergo filter sterilization (0.2 ?m) without loss of activity.

[0245] To address this issue, the inventors optimized a titration method to prepare polyplexes with a size of <100 nm. Titrating saRNA solutions (800 ?L, 1.00?10.sup.?3 mg mL.sup.?1) into polymer solutions (200 ?L, 0.18 mg mL.sup.?1) at a flow rate of 160 ?L min.sup.?1 yields smaller nanoparticles with a hydrodynamic diameter of ?70 nm, narrow dispersity (0.2) and high surface charge (+23 mV) (FIG. 4a). Increasing the weight ratio from 45:1 to 60:1 did not influence particle sizes. The inventors found that a hydrodynamic diameter of ?70 nm is sufficiently small enough for sterile filtration without losing any nanoparticles. The saRNA recovery after sterile filtration was monitored using Nanodrop at 260 nm (FIG. 4b). RNA concentrations before (black) and after (red) filtration were identical as was the hydrodynamic diameter (FIG. 4c), suggesting that no saRNA loss during sterile filtration

[0246] In order to demonstrate that polyplexes formed by the titration method enable high transfection efficiency even after sterile filtration, the inventors evaluated sterile filtered particles in vitro and in vivo. Polyplexes prepared using direct mixing were used as the control. While the transfection efficiency of polyplexes formed by direct mixing decreased by at least one order of magnitude (FIG. 4d) after sterile filtration, the titrated polyplexes were not affected at all (FIG. 4e), suggesting that nanoparticles of ?70 nm and narrow dispersity are favorable for sterile filtration. A similar phenomenon was also observed in vivo; polyplexes formed via titration had high luciferase expression (?10.sup.6 p/s) both before and after sterile filtration, while the ones generated by direct mixing had slightly lower luciferase expression (?10.sup.5 p/s) and were no longer effective after sterile filtration (FIGS. 4f and 4g). Thus the inventors concluded that the titration method enables reproducible and scalable production of pABOL/saRNA polyplexes that facilitate protein expression both in vitro and in vivo.

Example 4Increasing pABOL Molar Mass Enhances Luciferase Expression In Vivo

[0247] The inventors further investigated whether increasing the molar mass of pABOLs enhanced the delivery and expression of saRNA in vivo, using fLuc as a reporter protein (FIG. 5). They tested a range of pABOL molecular weights, from 8 to 167 kg mol.sup.?1, as these were the polymers that they found to effectively complex and condense saRNA (FIGS. 2b and 2c). Mice were injected with 5 ?g of fLuc saRNA/pABOL polyplexes prepared at ratio of 45:1 (w/w) either intramuscularly (IM) or intradermally (ID) and imaged after 7 d, which has been previously shown to be peak protein expression for Venezuelan Equine encephalitis virus (VEEV)..sup.13 The inventors used two commercially available linear PEIs as a positive controlPEI MAX, which was used in all of their transfection experiments, and in vivo jetPEI?, which has previously been show to more effectively deliver RNA in vivo,.sup.32 The inventors observed signal from only one leg of one mouse in the PEI MAX IM group, whereas the average of the jetPEI group was 8?10.sup.4 p/s (FIG. 5a), confirming that jetPEI is a more effective in vivo delivery agent for RNA. The inventors observed similar superiority of pABOL polyplexes in vivo as for the in vitro experiments; the 8, 41, 72, 100 and 167 kg mol.sup.?1 pABOLs had average luciferase expression of 5?10.sup.6 p/s when injected IM, ?62-fold higher than the jetPEI group. Interestingly, the 25 kg mol.sup.?1 pABOL had equivalent luciferase expression (?10.sup.5 p/s) to jetPEI when injected IM, resulting in parabolic relationship between pABOL molar mass and luciferase expression in vivo. The 8, 100 and 167 kg mol.sup.?1 groups had statistically significantly higher luciferase expression than the jetPEI when injected IM, with p=0.0446, 0.0332 and 0.0354, respectively. There was no signal from the ID jetPEI group (FIG. 5b). However, the pABOL ID mice had similarly high luciferase expression to the IM groups and the inventors again observed a parabolic relationship between expression and molar mass. The 8, 25 and 100 kg mol.sup.?1 groups had luciferase expression of ?10.sup.6 p/s, while the 41 and 72 kg mol.sup.?1 groups had luciferase expression of ?10.sup.5 p/s. However, only the 25 and 100 kg mol.sup.?1 groups were statistically significantly higher, with p=0.0093 and 0.0186, respectively. Without wishing to be bound by theory, the inventors postulate that this parabolic relationship is governed by a mechanism wherein high luciferase expression from the 8 kg mol.sup.?1 pABOL polyplexes results from more rapid reduction and thus rapid uptake of RNA in vivo, whereas the higher molar mass polymers are reduced less quickly but provide more adequate protection for the RNA potentially resulting high intracellular RNA delivery. Thus, the pABOLs with moderate molar mass (25 and 41 kg mol.sup.?1), which theoretically are reduced less quickly but provide less adequate RNA protection result in lower signal and more variability of protein expression in vivo.

[0248] Finally, the inventors sought to determine the optimal ratio of pABOL to saRNA in vivo (FIG. 5e,f). They used ratios of 1:1 to 60:1 with pABOL-100, and observed that ratios of <25:1 yielded luciferase expression of 104 p/s or less. However, with a ratio of 45:1 the luciferase expression increased to ?10.sup.6 p/s and there was no added benefit of increasing the ratio to 60:1. The inventors are the first to observe that increasing the molar mass of a cationic, bioreducible polymer enhances protein expression from saRNA in vivo.

Example 5pABOL Enhances the Quantity of Cells Expressing saRNA Both In Vivo and Ex Vivo in Human Skin Explants

[0249] After observing efficient saRNA delivery in vivo, the inventors then sought to investigate whether pABOLs enhance the quality or quantity of cells expressing saRNA both ex vivo in a clinically relevant human skin explant model and in vivo in mouse muscle and skin. For the skin explants, the inventors compared saRNA alone, the commercially available PEIs (PEI MAX and jetPEI) and 25, 72 and 100 kg mol.sup.?1 pABOL complexed with 2 ?g of enhanced green fluorescent protein (eGFP) saRNA (FIG. 6a,d). RNA alone resulted in eGFP expression in ?1% of human skin cells (FIG. 6a), and complexation with PEI MAX and jetPEI did not increase the number of eGFP-positive cells. However, the 25 kg mol.sup.?1 pABOL complexed with saRNA at a ratio of 10:1, 25:1 and 45:1 (w/w) increased the number of eGFP-positive cells to 3% and was statistically significantly higher than the RNA alone (p=0.0080, 0.0056 and 0.0457, respectively). Interestingly, the 72 kg mol.sup.?1 pABOL did not increase the number of eGFP-positive cells at any of the ratios tested, but the 100 kg mol.sup.?1 pABOL resulted in 1.5% and 2.5% eGFP-positive cells at ratios of 25:1 and 45:1 (p=0.0346 and 0.0170, respectively).

[0250] The inventors then sought to characterize whether the formulations were enhancing the amount of protein expression per cell as evidenced by quantifying the median fluorescence intensity (MFI) (FIG. 6d). RNA alone had an eGFP MFI of ?10.sup.2, and none of the formulations enhanced the protein expression per cell, which would manifest as a shift to the right on the x-axis of the histogram plot in FIG. 6d. It is hypothesized that this is due to the self-replicating nature of the VEEV vector, wherein upon entering a cell it exhausts the cellular translational machinery resulting in the maximum MFI per cell.

[0251] The inventors then tested whether increasing the molar mass of pABOL enhanced the number of cells expressing eGFP after IM and ID injection in mice (FIG. 6b,c). The inventors observed that RNA alone yielded expression in ?10% of cells when injected IM, which was only enhanced to ?20% of cells by 41 and 100 kg mol.sup.?1 pABOL (p=0.0074 and 0.0022, respectively). 8 kg mol.sup.?1 pABOL enhanced the eGFP+ cells to 16%, but this was not statistically significant. Similarly, the inventors observed that RNA alone yielded eGFP expression in 20% of cells after ID injection, which was increased to ?30% of cells with 8 and 100 kg mol.sup.?1 pABOL, although they were not statistically significant. jetPEI and 41 kg mol.sup.?1 pABOL did not enhance the number of eGFP+ cells when injected ID. The inventors postulate that this is due to differential cell types between the muscle and the skin, which may have different kinetics of pABOL reduction and saRNA expression. Similar to the human skin explants, there was no significant shift in GFP MFI (FIG. 6e,f), further indicating that the total protein expression relies on the number of cells and not the amount of protein being expressed by each cell. These results directly reflect the relationship between pABOL molar mass and luciferase expression in vivo demonstrated in FIG. 5.

[0252] While the 25 kg mol.sup.?1 pABOL resulted in ?4% of eGFP-positive cells in human skin explants, this is a relatively low transfection efficiency. While this strongly agrees with the in vivo RNA expression levels that Liang et al. observed after intramuscular and intradermal mRNA injection in rhesus macaques,.sup.33 the inventors sought to determine whether this correlated with luciferase expression in human skin explants. The inventors injected human skin explants with 2 ?g of fLuc saRNA complexed with jetPEI, 25, 72 or 100 kg mol.sup.?1 pABOL at a mass ratio of 45:1 (w/w). Indeed the 25 and 100 kg mol.sup.?1 pABOL polyplexes had the highest luciferase expression (100,000 p/s), which directly reflect the percentage of eGFP+ cells in FIG. 5a. Overall, the inventors found that pABOL enhances the percentage of cells expressing saRNA compared to RNA alone or commercially available PEIs, when injected IM or ID in vivo in mice or ID ex vivo in human skin explants.

Example 6pABOL-Delivered saRNA is Preferentially Expressed by Epithelial Cells in Human Skin Explants

[0253] The inventors then further investigated which cells in human skin explants were expressing eGFP saRNA after intradermal injection (FIG. 7). The inventors observed that human skin explants are composed primarily of epithelial cells (53.7%), dendritic cells (14.8%), fibroblasts (11.6%) and Langerhans cells (10.8%) (FIG. 7a). The remaining 9% is composed of more rare immune cells, including leukocytes (4.0%), natural killer (NK) cells (2.6%), T cells (1.7%), B cells (0.6%) and monocytes (0.2%). Despite the predominance of epithelial cells in the skin when injected alone, saRNA was expressed in dendritic cells (DCs) (18.5%), leukocytes (16.3%), fibroblasts (16.0%), epithelial cells (13.1%), B cells (12.7%), Langerhans cells (12.3%), monocytes (8.0%), T cells (1.6%) and NK cells (1.4%) (FIG. 7b). There was a similar trend in cell types for both PEI MAX and jetPEI formulations. However, for all of the pABOL polyplexes, epithelial cells were the dominant cell type expressing the saRNA (18?24%), followed by DCs, leukocytes, Langerhans cells, B cells, fibroblasts, monocytes, NK cells and T cells. The inventors hypothesize that the predominant uptake of RNA alone and PEIs by mostly immune cells indicates that these formulations are scavenged by professional immune cells, whereas the pABOL formulations may actively enhance cellular uptake into epithelial cells. These findings are similar to characterization by Liang et al. of which cells express mRNA encapsulated in lipid nanoparticle formulations when injected intradermally in rhesus macaques..sup.33 In that study they did not characterize the same cell types but found that the formulations were mostly taken up by and expressed in monocytes and DCs. It is unknown whether the total number or the phenotype of cells that express saRNA affect the immunogenicity of a vaccine. However, here the inventors show that formulating saRNA with pABOL results in uptake by a more diverse array of human skin cells compared to RNA alone or commercially available PEIs.

Example 7Hemagluttinin (HA) saRNA/pABOL Polyplexes Induce High HA Antibody Titers and Confer Complete Protection Against Flu Challenge In Vivo

[0254] The inventors then sought to assess the immunogenicity and protective capacity of HA-encoding saRNA delivered by pABOL, when injected either IM or ID (FIG. 8). Mice receive a prime and boost of either 1 or 0.1 ?g of saRNA complexed with either jetPEI, 8 kg mol.sup.?1 pABOL or 100 kg mol.sup.?1 pABOL at a ratio of 45:1 (w/w). The boost was administered 6 weeks after the initial prime. The mice were challenged IN with Cal/09 flu virus three weeks after the boost and weighed daily to monitor disease progression.

[0255] The na?ve mice in both the IM and ID groups all lost >25% of their body weight between days 4-6 and had to be culled according to the challenge protocol (FIG. 8a,b). In the IM injection groups, all mice in the PEI and 8 kg mol.sup.?1 pABOL groups were completely protected, even in the 0.1 ?g groups, with the 1 ?g 8 kg mol.sup.?1 pABOL group showing the least amount of weight loss at peak viremia (?8%). All the mice in the 1 ?g 100 kg mol.sup.?1 pABOL group were completely protected, but two mice in the 0.1 ?g 100 kg mol.sup.?1 pABOL group reached 25% weight loss on day 5 and had to be culled, thus resulting in 60% survival in this group. The HA IgG antibody titers (FIG. 8b) directly reflect the challenge results; all groups show increasing antibody titers between 3 and 6 weeks, and then after the boost. The 1 ?g 8 kDa pABOL group had the highest antibody titers (?40,000 ng/mL) after 9 weeks, whereas the PEI and 100 kg mol.sup.?1 pABOL groups that received 1 ?g were equivalent (?10,000 ng/mL). Even after 9 weeks, the 0.1 ?g 100 kg mol.sup.?1 pABOL group only reached a titer of ?100 ng/mL, whereas the 8 kg mol.sup.?1 pABOL and PEI groups reached titers of ?8,000 and ?2,000 ng/mL, respectively.

[0256] Compared to the IM injections, the ID injection groups were less protective against influenza challenge. Only the 1 ?g PEI group conferred complete protection, and resulted in ?12% weight loss during peak viremia. The 1 ?g 8 kg mol.sup.?1 pABOL had ?20% weight loss after 5 days and only reached antibody titers of ?500 ng/mL. The 0.1 ?g PEI, 0.1 ?g 8 kg mol.sup.?1 pABOL and 1/0.1 ?g 100 kg mol.sup.?1 pABOL groups all had approximately equivalent antibody titers, never reaching more than ?100 ng/mL and exhibiting low survival.

[0257] Overall, the 8 kg mol.sup.?1 pABOL group exhibited the highest antibody levels against HA after IM injection and conferred complete protection against flu challenge, even at a dose of only 0.1 ?g. These results show that route of administration (IM vs. ID) greatly influences the immunogenicity of polyplex-based vaccines, and that protein expression levels are not necessarily predictive of immunogenicity.

Materials

[0258] All solvents and reagents were obtained from commercial sources (Aldrich and Fisher) and used as received unless stated otherwise. Dialysis tubing (14 kg mol.sup.?1 molecular weight cut-off) was obtained from BioDesign Inc. of New York. Syringe filters with hydrophilic PVDF membrane were purchased from Sigma Aldrich.

Methods

Characterization

[0259] SEC: The molecular weights and dispersities were characterized using an Agilent PL GPC-50 instrument, equipped with a refractive index (RI) detector, with HPLC grade DMF (containing 0.075 wt % LiBr) as the eluent at a flow rate of 1.0 mL min.sup.?1 at 40? C. Two GRAM Linear columns were used in series. Near monodispersed poly(methyl methacrylate) standards were used to calibrate the instrument. The poly(amino amide)s were dissolved in HPLC grade DMF, containing 0.075 wt % LiBr, and filtered through 0.2 ?m syringe filters prior to analysis. Crude polymers were used for SEC characterization unless stated otherwise.

[0260] NMR: .sup.1H, .sup.13C{.sup.1H}, .sup.1H.sup.1H COSY and HSQC NMR spectra were recorded using a Bruker AV 400 MHz spectrometer at room temperature.

[0261] DLS: Dynamic light scattering was used to determine the hydrodynamic diameter (Dh) and polydispersity of the nanostructures formed between PABOLs and saRNA, in buffer solutions (20 Mm HEPES, 5 wt % glucose in water, pH 7.4), and was measured using the Zetasizer Nano ZS instrument. The scattering angle was fixed at 173?. Data processing was carried out using cumulant analysis of the experimental correlation function and the Stokes-Einstein equation was used to calculate the hydrodynamic radii. All solutions were analyzed using disposable polystyrene cuvettes.

[0262] Zeta potential: Zeta potential measurements were also conducted at 25? C. using a ZETASIZER Nano ZS instrument.

[0263] Nanodrop: The saRNA recovery was monitored using a Nanodrop One (Thermo Fisher) before and after sterile filtration of the polyplexes, through a 0.2 m syringe filter (membrane material: hydrophilic PVDF).

[0264] Mass spectroscopy: Mass spec characterizations were conducted using a Waters LCT Premier Mass Spectrometer. Samples were ionized using the electrospray (ES) technique.

[0265] TEM: Transmission electron microscopy (TEM) was performed on polyplexes that were prepared in H.sub.2O. 10 ?L of sample was pipetted directly onto a holey carbon film grid with 300 mesh copper (Agar Scientific, UK) and stained with 2% uranyl acetate, washed twice with DI H.sub.2O and allowed to air dry. Samples were then imaged on a TEM-2100 Plus Electron Microscope (JEOL USA, Peabody, MA, USA) using a voltage of 80 kV.

Improved Synthesis Procedure of PABOLs.

[0266] PABOL was synthesized by aza-Michael polyaddition of 4-amino-1-butanol (ABOL) to N,N-cystaminebisacrylamide (CBA). In a typical experiment, CBA (221.0 mg, 0.848 mmol), ABOL (78 ?L, 0.840 mmol) and trimethylamine (12 ?L, 0.084 mmol) were added into an ampoule flask charged with a stir bar. A mixed solvent, MeOH/water (176 ?L, 4/1, v/v), was also added into the ampoule flask. Polymerization was carried out in the dark at 45? C. under static nitrogen atmosphere. The reaction mixture became clear in less than 2 h. The mixture was allowed to react for 5 to 14 days (depending on the targeted molecular weight) to yield a highly viscous solution. Aliquots were taken at predetermined time intervals for .sup.1H NMR and SEC to monitor the conversion and molar mass. The reaction was stopped by MeOH dilution (50 mL) once the targeted molar mass was reached. The diluted reaction mixture was then acidified with 1.0 M HCl to pH ?4, and then purified by dialysis against acidic water (4.0 L, pH ?5, refreshed 6 times in 3 days). The polymers in their HCl-salt form were collected as white solid after freeze-dry.

Synthesis of Fluorescence Labelled PABOL-100.

[0267] PABOL-100 (30 mg; 2.857?10.sup.?4 mmol) was dissolved in DMF (400 ?L) in a vial charged with a stir bar, and 10 ?L of TEA was added to promote the dissolution of PABOL chains. Then, NIR 797 isothiocyanate (1 mg, 1.140?10.sup.?3 mmol) was added into the polymer solution. The molar ratio of [OH]/[isothiocyanate]=1/3.9. The mixture was allowed to react at 25? C. in dark for 18 h. The reaction mixture was then dialyzed in dark against dialysis against acidic water (500 mL, pH ?5, refreshed 6 times in 3 days). The labelled PABOL-100 were collected as dark green solid after freeze-dry. The graft density was calculated to be 9.75% (1 label per 10.26 OH) based on .sup.1H NMR spectrum.

Protocols of PABOL Reduction in the Presence of GSH.

[0268] PABOL and GSH were dissolved in D.sub.2O in a vial charged with a stir bar. The molar ratio of [SS]/[GSH]=1/10. The mixture was allowed to react at 37? C. for 24 h. Aliquots were taken at predetermined time intervals for .sup.1H NMR and Mass spectroscopy to determine the conversion. After the complete reduction of the disulfide bond, HPLC technique was employed to separate GSH, GSSG and the degradation products, using mixed solvent of MeCN (with 0.1% TFA) and H.sub.2O (with 0.1% TFA) at a flow rate of 10 mL/min using a Shimadzu HPLC instrument. The MeCN content of the mixed solvent increased gradiently from 5% to 30%.

In Vitro Transcription of saRNA.

[0269] Self-amplifying RNA derived from the Venezuelan Equine encephalitis Virus (VEEV) encoding firefly luciferase (fLuc), enhanced green fluorescent protein (eGFP) or hemagglutinin (HA) from the H1N1 A/California/07/2009 strain was produced using in vitro transcription. pDNA was transformed into E. coli and cultured in 50 mL of LB with 100 ?g/mL carbenicillin (Sigma Aldrich, UK) and isolated using a Plasmid Plux MaxiPrep kit (QIAGEN, UK). pDNA concentration and purity was measured on a NanoDrop One (ThermoFisher, UK) and subsequently linearized using MluI for 2 h at 37? C. and heat inactivated at 80? C. for 20 min. For in vitro transfections, capped RNA was synthesized using 1 ?g of linearized DNA template in a mMessage mMachine? reaction (Promega, UK) and purified using a MEGAClear? column (Promega, UK) according to the manufacturer's protocol. For in vivo experiments, uncapped in vitro RNA transcripts were synthesized using 1 ?g of linearized DNA template in a MEGAScript? reaction (Promega, UK) according to the manufacturer's protocol. Transcripts were then purified by overnight LiCl precipitation at ?20? C., pelleted by centrifugation at 14,000 rpm for 20 min, washed 1? with 70% EtOH, centrifuged at 14,000 rpm for 5 min, and then resuspended in UltraPure H.sub.2O. Purified transcripts were then capped using the ScriptCap? m7G Capping System (CellScript, Madison, WI, USA) and ScriptCapt? 2-O-Methyltransferase Kit (CellScript, Madison, WI, USA) simultaneously according to the manufacturer's protocol. Capped transcripts were then purified by LiCl precipitation as detailed above, resuspended in UltraPure H.sub.2O and stored at ?80? C. until further use.

Polyplex Formation Between PABOL and saRNA.

[0270] Stock solutions of PEI, PABOLs and saRNA were prepared first by directly dissolving these materials in molecular grade water and stored in fridge. The concentration of the stock solutions are 2.00 ?g/L (PEI), 0.24 ?g/L (fLuc Mut RepRNA) and 5.00 ?g/?L (PABOLs, in vitro studies) or 50 ?g/?L (PABOLs, in vivo studies), respectively. Polyplexes were prepared using two methods: a) direct mixing and b) titration.

[0271] a) Direct mixing: In a typical procedure, 4.17 ?L of the saRNA stock solution was diluted to 200 ?L using the HEPES buffer (20 mM HEPES, 5 wt % glucose in water, pH 7.4). A predetermined amount of polymer stock solution was also diluted to 800 ?L using the same buffer. Each tube was vortexed and centrifuged to ensure the homogeneity. Then, the polymer buffer solution was added to the saRNA buffer solution rapidly, following with vortex for 20 s to form the complex. A series of complex solutions were prepared with the polymer/saRNA weight ratio ranging from 1/1 to 60/1.

[0272] b) Titration: In a typical procedure, 4.17 ?L of the saRNA stock solution was diluted to 800 ?L using the HEPES buffer (20 mM HEPES, 5 wt % glucose in water, pH 7.4). A predetermined amount of polymer stock solution was also diluted to 200 ?L using the same buffer in centrifuge tubes, equipped with stir bars. Each tube was placed on a stir plate and stirred at 1200 rpm at ambient temperature. Then, the RNA solution was added to the polymer solution at a rate of 160 ?L/min (unless otherwise stated). A series of complex solutions were prepared with the polymer/saRNA weight ratios ranging from 1/1 to 60/1.

Protocols for In Vitro Transfection Studies.

[0273] Transfections were performed in HEK293T.17 cells (ATCC, USA) that were maintained in culture in complete Dulbecco's Modified Eagle's Medium (cDMEM) (Gibco, Thermo Fisher, UK) containing 10% fetal calf serum (FCS), 5 mg/mL L-glutamine and 5 mg/mL penicillin/streptomycin (Thermo Fisher, UK). Cells were plated at a density of 50,000 cells per well in a clear 96 well plate 24 h prior to transfection. For the transfection, the media was completely removed and replace with 50 ?L of pre-warmed transfection medium (DMEM with 5 mg/mL L-glutamine). 100 ?L of the polyplex solution was added to each well and allowed to incubate for four hours, then the transfection media was completely removed and replaced with 100 ?L of cDMEM. After 24 h from the initial transfection, 50 ?L of media was removed from each well and 50 ?L of ONE-Glo? D-luciferin substrate (Promega, UK) was added and mixed well by pipetting. The total volume was transferred to a white 96-well plate (Costar) and analyzed on a FLUOstar Omega plate reader (BMG LABTECH, UK) and background from the media control wells was subtracted. For the glutathione inhibition assay, cells were incubated with 200 ?M buthionine sulfoximine (BSO), a known glutathione inhibitor,.sup.34 for 4 hours prior to the transfection; and then the transfection was performed as detailed above.

Cytotoxicity of Polyplexes.

[0274] For analysis of polyplex cytotoxicity, cells were transfected with varying ratios of PABOL and PEI to saRNA ranging from 10:1 to 450:1 (w/w) according to the above protocol. 24 h after the initial transfection, 20 ?L of CellTiter-Blue reagent (Promega, UK) was added to each well and allowed to incubate for 1 h. The plate was then analyzed for absorbance on a FLUOstar Omega plate reader (BMG LABTECH, UK) and normalized to the media control.

In Vivo fLuciferase Expression in Mice.

[0275] All animals were handled in accordance with the UK Home Office Animals Scientific Procedures Act 1986 and with an internal ethics board and UK government approved project and personal license. Food and water were supplied ad libitum. Female BALB/c mice (Charles River, UK) 6-8 weeks of age were placed into groups (n=5) and housed in a fully acclimatized room. Mice were injected intramuscularly (IM) in both hind legs or intradermally (ID) with 5 ?g of fLuc saRNA in a total volume of 50 ?L. After 7 days, the mice were injected intraperitoneally (IP) with 100 ?L of XenoLight RediJect D-Luciferin Substrate (Perkin Elmer, UK) and allowed to rest for 10 min. Mice were then anesthetized using isoflurane and imaged on an In Vivo Imaging System (IVIS) FX Pro (Kodak Co., Rochester, NY, USA) equipped with Molecular Imaging Software Version 5.0 (Carestream Health, USA) for 2 min. Signal from each injection site was quantified using Molecular Imaging software and expressed as relative light units (p/s).

Flow Cytometry Analysis of eGFP Expression in Human Skin Explants.

[0276] Surgically resected specimens of human skin tissue were collected at Charing Cross Hospital, Imperial NHS Trust, London, UK. All tissues were collected after receiving signed informed consent from patients, under protocols approved by the Local Research Ethics Committee. The tissue was obtained from patients undergoing elective abdominoplasty or mastectomy surgeries. Tissue was refrigerated until arrival in the laboratory where the subcutaneous layer of fat was removed, and the tissue was excised into 1 cm.sup.2 sections. Explants were incubated at 37? C. with 5% CO.sub.2 in petri dishes with 10 mL of cDMEM. Media was replaced daily. Explants were injected intradermally (ID) using a Micro-Fine Demi 0.3 mL syringe (Becton Dickinson, UK) with 2 ?g of eGFP saRNA/pABOL polyplexes in a volume of 100 ?L. After three days, skin explants were minced well with scissors and incubated in 3 mL DMEM supplemented with 1 mg/mL collagenase P (Sigma, UK) and 5 mg/mL dispase II (Sigma, UK) for 4 h at 37? C. on a rotational shaker. Digests were then filtered through a 70 ?m cell strainer and centrifuged at 1750 RPM for 5 min. Cells were then resuspended in 1 mL of FACS buffer (PBS+2.5.% FCS) at a concentration of 1E7 cells/mL. 100 ?L of cell suspension was added to a FACS tube and stained with Fixable Aqua Live/Dead Cell stain (Thermo Fisher, UK) dilution 1:400 in FACS buffer for 20 min on ice. Cells were then washed with 2.5 mL of FACS buffer, centrifuged at 1750 rpm for 5 min and stained with a panel of antibody to identify each cell type, as described in Supplementary Table 1, for 30 min. Cells were then washed with 1 mL of FACS buffer, centrifuged at 1750 rpm for 5 min and resuspended in 250 ?L PBS. Cells were fixed by addition of 250 ?L of 3.0% paraformaldehyde for a final concentration of 1.5%, and refrigerated until flow cytometry analysis. Samples were analyzed on a LSRFortessa? (BD Biosciences, UK) with FACSDiva software (BD Biosciences, UK) with 100,000 acquired live cell events. Gating was performed as previously described..sup.35 Phenotypic identity of GFP-positive cells was quantified using FlowJo Version 10 (FlowJo LLC, Oregon, USA).

Flow Cytometry Analysis of eGFP Expression in Murine Skin and Muscle.

[0277] Female BALB/c mice (Charles River, UK) 6-8 weeks of age were placed into groups (n=5) and housed in a fully acclimatized room. Mice were injected intramuscularly (IM) in both hind legs or intradermally (ID) with 5 ?g of eGFP saRNA in a total volume of 50 ?L. After 7 days, the mice were culled and the muscle or skin around the injection site was excised and put in 3 mL DMEM supplemented with 1 mg/mL collagenase P (Sigma, UK) and 5 mg/mL dispase II (Sigma, UK) for 4 h at 37? C. on a rotational shaker. Digests were then filtered through a 70 ?m cell strainer and centrifuged at 1750 RPM for 5 min. Cells were then resuspended in 1 mL of FACS buffer (PBS+2.5.% FCS) at a concentration of 1E7 cells/mL. 100 ?L of cell suspension was added to a FACS tube and stained with Fixable Aqua Live/Dead Cell stain (Thermo Fisher, UK) dilution 1:400 in FACS buffer for 20 min on ice. Cells were then washed with 1 mL of FACS buffer, centrifuged at 1750 rpm for 5 min and resuspended in 250 ?L PBS. Cells were fixed by addition of 250 ?L of 3.0% paraformaldehyde for a final concentration of 1.5%, and refrigerated until flow cytometry analysis. Samples were analyzed on a LSRFortessa? (BD Biosciences, UK) with FACSDiva software (BD Biosciences, UK) with 100,000 acquired live cell events. Phenotypic identity of GFP-positive cells was quantified using FlowJo Version 10 (FlowJo LLC, Oregon, USA).

Ex Vivo fLuciferase Expression in Human Skin Explants.

[0278] Human skin tissue was collected and excised as described above. Explants were incubated at 37? C. with 5% CO.sub.2 in petri dishes with 10 mL of cDMEM. Media was replaced daily. Explants were injected intradermally (ID) using a Micro-Fine Demi 0.3 mL syringe (Becton Dickinson, UK) with 2 ?g of fLuc saRNA/pABOL polyplexes in a volume of 100 ?L. After three days, skin explants were inverted and the media was replaced with 5 mL of cDMEM supplemented with 100 ?L of XenoLight RediJect D-Luciferin Substrate (Perkin Elmer, UK) and imaged on an In Vivo Imaging System (IVIS) FX Pro (Kodak Co., Rochester, NY, USA) equipped with Molecular Imaging Software Version 5.0 (Carestream Health, USA) for 60 min. Signal from each injection site was quantified using Molecular Imaging software and expressed as relative light units (p/s).

In Vivo Immunogenicity of HA saRNA.

[0279] BALB/c mice were immunized IM in one hind leg with either 1 or 0.1 ?g of HA saRNA formulated with either in vivo jet-PEI?, PABOL-8 (Table 1, #2) or PABOL-100 (Table 1, #8) in a total volume of 50 ?L, and boosted after 6 weeks. Blood was collected after 3, 6 and 9 weeks from study onset via tail bleeding, centrifuged at 10,000 rpm for 5 min and then the serum was removed and stored at ?80? C. until further use.

HA-Specific ELISA.

[0280] A semi-quantitative immunoglobulin ELISA protocol was performed as previously described..sup.36 Briefly, 0.5 ?g/mL of HA coated ELISA plates were blocked with 1% BSA/0.05% Tween-20 in PBS. After washing, diluted samples were added to the plates and incubated for 2 h, washed, and a 1:4,000 dilution of anti-mouse IgG-HRP (Southern Biotech, UK) was used. Standards were prepared by coating ELISA plate wells with anti-mouse Kappa (1:1,000) and Lambda (1:1,000) light chain (Serotec, UK), blocking with PBS/1% BSA/0.05% Tween-20, washing and adding purified IgG (Southern Biotech, UK) starting at 1,000 ng/mL and titrating down with a 5-fold dilution series. Samples and standard were developed using TMB (3,3-5,5-tetramethylbenzidine) and the reaction was stopped after 5 min with Stop solution (Insight Biotechnologies, UK). Absorbance was read on a spectrophotometer (VersaMax, Molecular Devices) with SoftMax Pro GxP v5 software.

Influenza Challenge.

[0281] 3 weeks after the boost injection, mice were challenge with XXX pfu of influenza (Cal/09) suspending in 100 uL of PBS. Mice were anesthetized using isoflurane, challenged intranasally (IN), and weighed each day to determine weight loss. According to challenge protocol, mice were culled if they sustained more than three days of 20% weight loss or one day of 75% weight loss.

Statistical Analysis.

[0282] Graphs and statistics were prepared in GraphPad Prism, version 8. Statistical differences were analyzed using either a two-tailed t test or an ordinary one-way ANOVA with multiple comparisons, with ?=0.05 used to indicate significance.

REFERENCES

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