Data for: "Delivery of self-amplifying mRNA vaccines by cationic lipid nanoparticles: The impact of cationic lipid selection"

Self-amplifying RNA (SAM) represents a versatile tool that can be used to develop potent vaccines, potentially able to elicit strong antigen-specific humoral and cellular-mediated immune responses to virtually any infectious disease. To protect the SAM from degradation and achieve efficient delivery, lipid nanoparticles (LNPs), particularly those based on ionizable amino-lipids, are commonly adopted. Herein, we compared commonly available cationic lipids, which have been broadly used in clinical investigations, as an alternative to ionizable lipids. To this end, a SAM vaccine encoding the rabies virus glycoprotein (RVG) was used. The cationic lipids investigated including 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), dimethyldioctadecylammonium (DDA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP), 1,2-stearoyl-3-trimethylammonium-propane (DSTAP) and N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ). Whilst all cationic LNP (cLNP) formulations promoted high association with cells in vitro, those formulations containing the fusogenic lipid 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE) in combination with DOTAP or DDA were the most efficient at inducing antigen expression. Therefore, DOTAP and DDA formulations were selected for further in vivo studies and were compared to benchmark ionizable LNPs (iLNPs). Biodistribution studies revealed that DDA-cLNPs remained longer at the injection site compared with DOTAP-cLNPs and iLNPs when administered intramuscularly in mice. However, both the cLNP formulations and the iLNPs induced strong humoral and cellular-mediated immune responses in mice that were not significantly different at a 1.5 µg SAM dose. In summary, cLNPs based on DOTAP and DDA are an efficient alternative to iLNPs to deliver SAM vaccines. Table 1. Physicochemical characterization of SAM-LNPs produced by microfluidics. Formulations were composed of DOPE, a cationic lipid and DMG-PEG2000 at 49:49:2 molar ratio or DSPC, Chol, a cationic lipid/ionizable lipid and DMG-PEG2000 at 10:48:40:2 molar ratio. E.E. (encapsulation efficiency); ZP (zeta-potential). Results are represented as mean ± of three independent experiments. Figure 1. Cellular uptake RVG-SAM cLNPs and iLNPs in presence (A, B) and absence of 5% FBS (C, D) represented in terms of percentage of Dil-C18+ cells (A, C) and mean fluorescence intensity (B, D). cLNPs were composed of DOPE, a cationic (DOTAP, DDA, DC-Chol, DMTAP or DOBAQ) and DMG-PEG2000 at 49:49:2 mole % or DSPC, Chol, a cationic lipid and DMG-PEG2000 at 10:48:40:2 mole %. Results are represented as mean ± SD of three experiments. Statistical significance: P < 0.05 (*). Figure 2. In vitro potency of RVG-SAM iLNPs and DOPE-cLNPs in presence (A) and absence of 5% FBS (B). LF2000 (Lipofectamine2000). Results are represented as mean ± SD of three independent experiments. Figure 4. Total anti-RVG IgG titers in mice upon intramuscular injection of SAM formulated in cLNPs, DOTAP-CNE, iLNPs or the commercial vaccine Rabipur on days 0 and 28. Sera were collected after 27 (A) and 42 (B) days and total IgG titers were quantified using PLATELIA RABIES II KIT (Bio-Rad). Dots depict measurements from pools of 2 sera each. The solid lines represent the geometric mean titer (GMT) of each group (n=5). Dotted lines at 0.5 and 0.125 EU/mL correspond to protection threshold and limit of quantification respectively. HD (human dose). C) Frequencies of RVG-specific cytokine producing CD8+ (C) and CD4+ T cells (D) analyzed with Boolean gates. CD4+ T cells were represented as Th1 and Th0 cells according to secreted cytokines. T cell results are represented as mean ± SD of three replicates. For the statistical analysis of T cell responses, DOTAP-cLNPs, DDA-cLNPs and DOTAP-CNE were compared to iLNPs at the same SAM dose: non-significant (ns); p < 0.05 (*) Figure 5. Biodistribution of DOTAP-cLNPs, DDA-cLNPs and benchmark iLNPs in mice following intramuscular administration. A) Images acquired at relevant time points. B) Biodistribution pharmacokinetics. C) Calculated areas under the curve for each formulation. Results are represented as total flux in the region of interest, in pink, as mean ± SD of five animals per group. Statistical significance of DDA-cLNPs compared to DOTAP-cLNPs and iLNPs: p < 0.05 (*).

自扩增RNA（Self-amplifying RNA，SAM）是一类多功能工具，可用于开发强效疫苗，理论上可针对几乎所有传染病诱导强烈的抗原特异性体液免疫与细胞介导免疫应答。为保护SAM免受降解并实现高效递送，脂质纳米粒（lipid nanoparticles，LNPs）——尤其是基于可电离氨基脂质的LNPs——是目前常用的递送载体。本研究选取临床研究中广泛应用的商品化阳离子脂质作为可电离脂质的替代方案开展对比研究。为此，本研究采用编码狂犬病毒糖蛋白（rabies virus glycoprotein，RVG）的SAM疫苗作为模型。本次考察的阳离子脂质包括：3β-[N-(N',N'-二甲基氨基乙基)-氨基甲酰基]胆固醇（3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol，DC-Chol）、二甲基双十八烷基氯化铵（dimethyldioctadecylammonium，DDA）、1,2-二油酰基-3-三甲基铵丙烷（1,2-dioleoyl-3-trimethylammonium-propane，DOTAP）、1,2-二肉豆蔻酰基-3-三甲基铵丙烷（1,2-dimyristoyl-3-trimethylammonium-propane，DMTAP）、1,2-二硬脂酰基-3-三甲基铵丙烷（1,2-stearoyl-3-trimethylammonium-propane，DSTAP）以及N-(4-羧基苄基)-N,N-二甲基-2,3-双(油酰氧基)丙-1-铵盐（N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium，DOBAQ）。
尽管所有阳离子脂质纳米粒（cationic LNP，cLNP）制剂在体外均能实现较高的细胞结合率，但其中搭载融合性脂质1,2-二油酰基-sn-甘油-3-磷酸乙醇胺（1,2-dioleoyl-sn-3-phosphoethanolamine，DOPE）并联合DOTAP或DDA的制剂，诱导抗原表达的效率最为优异。因此，本研究选取DOTAP与DDA制剂开展后续体内研究，并将其与基准可电离脂质纳米粒（ionizable LNPs，iLNPs）进行对比。
体内分布研究结果显示，小鼠肌肉注射后，DDA-cLNPs在注射部位的滞留时长显著长于DOTAP-cLNPs与iLNPs。然而，在SAM给药剂量为1.5 μg时，两类cLNP制剂与iLNPs均可在小鼠体内诱导强烈的体液免疫与细胞介导免疫应答，且各组间应答水平无显著差异。
综上，基于DOTAP与DDA的cLNP可作为iLNPs的高效替代载体，用于SAM疫苗的递送。

表1 微流控法制备的SAM-LNPs理化表征。制剂组成分为两类：一类为DOPE、阳离子脂质与DMG-PEG2000按照49:49:2的摩尔比配制；另一类为DSPC、胆固醇、阳离子/可电离脂质与DMG-PEG2000按照10:48:40:2的摩尔比配制。E.E.（包封率，encapsulation efficiency）；ZP（Zeta电位，zeta-potential）。结果以三次独立实验的平均值±标准差表示。

图1 有无5%胎牛血清（FBS）时，RVG-SAM cLNP与iLNP的细胞摄取情况，分别以Dil-C18+细胞占比（A、C）与平均荧光强度（B、D）表征。cLNP制剂组成：一类为DOPE、阳离子脂质（DOTAP、DDA、DC-Chol、DMTAP或DOBAQ）与DMG-PEG2000按照49:49:2的摩尔百分比配制；另一类为DSPC、胆固醇、阳离子脂质与DMG-PEG2000按照10:48:40:2的摩尔百分比配制。结果以三次实验的平均值±标准差表示。统计学显著性：P < 0.05（*）。

图2 有无5% FBS时，RVG-SAM iLNPs与DOPE-cLNPs的体外活性（A、B）。LF2000即Lipofectamine2000。结果以三次独立实验的平均值±标准差表示。

图4 小鼠于第0天和第28天肌肉注射搭载于cLNPs、DOTAP-CNE、iLNPs或商业疫苗Rabipur的SAM制剂后，总抗RVG IgG滴度检测结果。分别于第27天（A）与第42天（B）采集血清，采用PLATELIA RABIES II KIT（Bio-Rad）定量总IgG滴度。每个数据点代表2份血清混合样本的检测结果；实线为每组的几何平均滴度（geometric mean titer，GMT），每组n=5。0.5 EU/mL与0.125 EU/mL的虚线分别对应保护阈值与定量限。HD为人体剂量（human dose）。C）通过布尔门分析RVG特异性细胞因子分泌型CD8+（C）与CD4+ T细胞（D）的频率。根据分泌的细胞因子，CD4+ T细胞可分为Th1型与Th0型。T细胞结果以三次重复实验的平均值±标准差表示。T细胞应答的统计学分析：将DOTAP-cLNPs、DDA-cLNPs与DOTAP-CNE与同剂量SAM的iLNPs组进行对比：无显著性差异（ns）；P < 0.05（*）。

图5 小鼠肌肉注射DOTAP-cLNPs、DDA-cLNPs与基准iLNPs后的体内分布情况。A）各时间点的成像图像；B）体内分布药代动力学曲线；C）各制剂的曲线下面积计算结果。结果以感兴趣区域（粉色标记）的总荧光通量表示，每组5只小鼠，数据为平均值±标准差。DDA-cLNPs与DOTAP-cLNPs、iLNPs组的统计学显著性对比：P < 0.05（*）。