key: cord-0699836-6osj0dwc
authors: Gerhardt, Alana; Voigt, Emily; Archer, Michelle; Reed, Sierra; Larson, Elise; Van Hoeven, Neal; Kramer, Ryan; Fox, Christopher; Casper, Corey
title: A Flexible, Thermostable Nanostructured Lipid Carrier Platform for RNA Vaccine Delivery
date: 2022-03-16
journal: Mol Ther Methods Clin Dev
DOI: 10.1016/j.omtm.2022.03.009
sha: f4621c37e8537d33e2b67b6f1da1fcbb1802d75c
doc_id: 699836
cord_uid: 6osj0dwc

Current RNA vaccines against SARS-CoV-2 are limited by instability of both the RNA and the lipid nanoparticle delivery system, requiring storage at -20°C or -70°C and compromising universally accessible vaccine distribution. This study demonstrates the thermostability and adaptability of a nanostructured lipid carrier (NLC) delivery system for RNA vaccines that has the potential to address these concerns. Liquid NLC alone is stable at refrigerated temperatures for ≥ 1 year, enabling stockpiling and rapid deployment by point-of-care mixing with any vaccine RNA. Alternatively, NLC complexed with RNA may be readily lyophilized and stored at room temperature for ≥ 8 months or refrigerated temperature for ≥ 21 months while still retaining the ability to express protein in vivo. The thermostability of this NLC/RNA vaccine delivery platform could significantly improve distribution of current and future pandemic response vaccines, particularly in low-resource settings.

accessible vaccine distribution. This study demonstrates the thermostability and adaptability of a 28 nanostructured lipid carrier (NLC) delivery system for RNA vaccines that has the potential to address 29 these concerns. Liquid NLC alone is stable at refrigerated temperatures for ≥ 1 year, enabling 30 stockpiling and rapid deployment by point-of-care mixing with any vaccine RNA. Alternatively, NLC 31 complexed with RNA may be readily lyophilized and stored at room temperature for ≥ 8 months or 32 refrigerated temperature for ≥ 21 months while still retaining the ability to express protein in vivo. The 33 thermostability of this NLC/RNA vaccine delivery platform could significantly improve distribution of 34 current and future pandemic response vaccines, particularly in low-resource settings. 70-100 nm diameter RNA/LNP complexes which protect the RNA from RNase degradation and allow 70 for successful endocytosis by the cell. 20, 21 However, stability of both the RNA and LNP remain an 71 issue, [9] [10] [11] with sensitivity to frozen temperatures resulting in detrimental impacts to their colloidal 72 stability after freeze/thaw. 22,23 A number of recent studies have reported on improvements to the long-73 term thermostability of RNA vaccines at non-frozen temperatures; 24-26 however, all currently authorized 74 RNA vaccines available in the United States still require frozen storage. 27,28 75 A number of alternative lipid-based delivery systems have been proposed and developed to 76 deliver RNA vaccines. [29] [30] [31] Here, we demonstrate the ability of a lyophilizable, thermostable 77 nanostructured lipid carrier (NLC) system to effectively deliver replicating RNA-based vaccines by 78 intramuscular injection. This NLC delivery system can also be complexed and lyophilized with mRNA. 79 The liquid NLC alone maintains stability for at least 1 year of storage at refrigerated temperatures while Long-term stability of lyophilized SEAP NLC/saRNA complexes 144 Finally, we demonstrate the long-term thermostability of the NLC-based RNA vaccine platform 145 using a self-amplifying RNA antigen expression reporter system expressing secreted alkaline 146 phosphatase (SEAP-saRNA) (Supplemental Figure S3B ), which allows for sensitive mouse serum 147 detection of i.m.-injected saRNA. Lyophilized SEAP NLC/saRNA complexes with 20% w/v sucrose as 148 a lyoprotectant stored at 4°C, 25°C, and 40°C are compared with frozen complexes stored at -80°C and -149 20C°, liquid complexes stored at 4°C and 25°C, and freshly made complexes prepared each analysis 150 day. All lyophilized samples maintain an elegant, white cake throughout the study with no discoloration 151 or cracking and minimal cake shrinkage. All lyophilized samples readily reconstitute with nuclease-free 152 water into the milky white solution typically observed for the NLC/RNA complexes ( Figure 4A ). 153 Initially, all NLC/saRNA complexes ( Figure 4B ) measure 125±10 nm in diameter, including 154 liquid, frozen, and lyophilized versions. Differences of less than 15% are observed between the initial 155 and final timepoints for all conditions except for frozen material stored at -20°C. This demonstrates the 156 excellent colloidal stability of NLC/RNA complexes, allowing them to withstand the stresses of the stored at -20°C, this did not impact the ability of the NLC/saRNA complex to drive protein expression 160 in vivo upon i.m. injection ( Figure 4D , 4E). 161 RNA integrity in the NLC/saRNA complexes is again retained after lyophilization and after 162 freeze/thaw as demonstrated by agarose gel electrophoresis after extraction of the RNA from the stored 163 NLC complexes, and this integrity is maintained after long-term storage ( Figure 4C Figure S4 , S5C). 173 The ability of stored NLC/saRNA to express protein in vivo is demonstrated by injection of 100 174 ng of NLC/SEAP-saRNA complex i.m. into C57BL/6 mice, followed by collection of mouse sera 5 days 175 post-injection and analysis of SEAP content by enzymatic assay ( Figure 4D ). At each timepoint, a group 176 receiving freshly prepared (i.e., not stored) complex was included as a positive control, and a group that 177 received an injection of a 10% sucrose solution was a negative control. SEAP expression at each 178 timepoint was normalized to this negative control in Figure 4D Figure 4D ). At the 21 month timepoint, the lyophilized 25°C stored and frozen -20°C stored 187 complexes show a decrease in SEAP expression relative to the freshly complexed material; however, 188 SEAP expression still remains significantly above baseline levels for both conditions ( Figure 4E ). A key 189 observation is that after 21 months of storage, no significant difference was detected in the level of in 190 vivo expressed SEAP for the lyophilized 4°C and frozen -80°C stored complexes when compared with demonstrate that a safe and effective NLC-based RNA vaccine delivery system 30 has potential to enable 206 greatly increased thermostability relative to current LNP formulations. The liquid NLC alone is stable at 207 refrigerated temperatures for greater than 1 year. NLC complexed with mRNA or saRNA is able to be 208 lyophilized with both lyophilized and frozen forms of SEAP NLC/saRNA showing stability after storage 209 for extended periods of time. Moreover, upon reconstitution, NLC-formulated RNA vaccine retains its 210 integrity by agarose gel electrophoresis for at least 2 weeks of storage at refrigerated temperatures. This 211 NLC-based delivery technology may have significant applications for RNA vaccine manufacture, 212 storage, distribution, and overall cost due to its thermostable properties. 213 We hypothesize multiple mechanisms behind the improved thermostability of NLC-based 214 delivery formulations relative to LNP-based formulations. First, the robust physical stability of the NLC 215 allows for minimal growth in particle size, retention of constituent components, and maintenance of Third and most importantly, the physical characteristics of this NLC-based RNA vaccine 232 formulation allow for lyophilization, a technique commonly used to stabilize vaccines and biologics and 233 eliminate a cold chain requirement. 7, 8, [33] [34] [35] [36] In lyophilized drug products, non-reducing sugars (such as 234 sucrose) act as lyoprotectants through multiple proposed mechanisms such as replacing water in 235 hydrogen bonding with the components of the system or enclosing the system within the rigid sugar 236 matrix of the dried state where enzymatic or other degradation is limited. 37 . As noted above, mRNA 237 stability appears to be the limiting factor in the shelf-life of current mRNA/LNP vaccines. To address 238 that concern, RNA molecules alone have been shown to be amenable to lyophilization with Jones et al. 239 reporting that lyophilized RNA retained its ability to drive protein expression after storage at ≤ 4°C for 240 up to 10 months. 34 However, lyophilization of liposome-like formulations has been pursued for decades 241 (reviewed by Franze, et al. 37 and Wang, et al. 38 ) but can be difficult due to the liposome's physical 242 structure (i.e., a lipid bilayer surrounding a core aqueous phase). The freezing, drying, and reconstitution 243 steps of lyophilization may result in bilayer rupture, drug leakage, and/or colloidal instability. 37,38 While 244 the exact structure of mRNA-loaded LNPs is still being investigated 39-41 and may vary based on 245 composition and production process, their hypothesized core-shell structure 10 may still be susceptible to 246 the same rupture, leakage, and instability as liposomes. Recent published attempts at RNA/LNP vaccine 247 lyophilization have been semi-successful, but either were not evaluated after long-term storage 22 or 248 showed significant loss of RNA activity after long-term storage even with the addition of 249 lyoprotectants. 23 While optimization of LNP lyophilization may yet be attempted (reviewed by Chen and 250 colleagues 42 ), the technical challenge of redesigning and clinically testing lyophilizable liposome-based 251 J o u r n a l P r e -p r o o f RNA vaccine delivery formulations is significant and without guaranteed success. In contrast, the 252 structure of the NLC delivery system is more similar to an oil-in-water emulsion than to an LNP. Bilayer 253 rupture and/or drug leakage are not a concern with this system because the RNA is complexed to the 254 surface of the NLC, and maintenance of RNA integrity and colloidal stability have been demonstrated in 255 our study. Furthermore, lyophilized vaccines containing squalene-based adjuvant systems have 256 previously demonstrated potential for long-term vaccine thermostability. 43 257 Another potential advantage of the NLC delivery system for pandemic response is its 258 straightforward and scalable manufacturing process. This employs similar processes and equipment as 259 oil-in-water emulsion technology already used in licensed vaccineskey manufacturing properties to 260 support large-scale pandemic response. The NLC system also does not require the use of specially 261 designed, proprietary ionizable lipids to generate an appropriate immune response as is the case with 262 LNP-based formulations. 44 Rather, the system relies on the presence of squalenein combination with 263 innate immune-stimulating dsRNA intermediates produced by saRNAsto stimulate robust immune 264 responses. Therefore, the NLC can use the commercially available cationic lipid DOTAP as the source 265 of positive charge. Furthermore, in contrast to currently authorized RNA/LNP vaccines, the NLC 266 delivery system is manufactured separately from the RNA. For pandemic preparedness, the long-term 267 refrigerator stable NLC alone could be stockpiled to enable rapid response. Because it is manufactured 268 separately, RNA of different lengths or with multiple genetic variations may be rapidly synthesized and 269 complexed on the outside of the NLC, allowing rapid vaccine adaptation to evolving viral variants or 270 emerging pathogens. 271 We do note that the presented long-term stability data of the lyophilized NLC system is with 272 RNA expressing a reporter protein (i.e., SEAP) rather than a vaccine antigen. While use of a reporter While the current study demonstrates the excellent thermostability of an NLC/RNA complex, 283 this was a proof-of-concept attempt for this system and did not include optimization of the formulation 284 matrix (i.e., buffer, pH, or additional excipients) or lyophilization cycle. Further work will be conducted 285 to optimize this system to push the limits of manufacturing, storage, and use conditions and then to 286 demonstrate its utility with an actual vaccine product. Future development of a spray-dried RNA 287 vaccine, for example, could potentially harness the advantages of a dried system in terms of stability as 288 seen in this study, while decreasing potential manufacturing bottlenecks that lyophilization can pose. 289 Additionally, further optimization of the NLC/RNA drug product formulation may also allow for greater 290 liquid stability leading to significantly easier manufacturing, storage, and distribution. Widespread (pT7-VEEV-SEAP-V2) reflects the same antibiotic resistance gene and subgenomic promoter changes 313 described above to allow for optimal comparison to pT7-VEE-Zika-prME in the vaccine 314 immunogenicity studies in Figure 2 . All plasmid sequences were confirmed using Sanger sequencing. 315 DNA templates were amplified in E. coli and isolated using maxi or gigaprep kits (Qiagen) and 316 linearized by NotI restriction digest (New England Biolabs). Linearized DNA was purified by phenol 317 chloroform extraction. 318 Generation of saRNA stocks was achieved by T7 promoter-mediated in vitro transcription using 321 NotI-linearized DNA template. In vitro transcription was performed using an in house-optimized 322 protocol using T7 polymerase, RNase inhibitor, and pyrophosphatase enzymes procured from Aldevron. 323 DNA plasmid was digested away (DNase I, Aldevron) and cap0 structures were added to the transcripts 324 by vaccinia capping enzyme, GTP, and S-adenosyl-methionine (Aldevron). RNA was then purified from 325 the transcription and capping reaction components by chromatography using a CaptoCore 700 resin (GE 326 Healthcare) followed by diafiltration and concentration using tangential flow filtration. The saRNA 327 material was terminally filtered with a 0.22µm polyethersulfone filter and stored at -80°C until use. All Scientific) was diluted in 10 mM sodium citrate trihydrate and also heated to 70°C in a bath sonicator. 337 After all components were dissolved, the oil and aqueous phases were mixed at 7,000 rpm in a high-338 speed laboratory emulsifier (Silverson Machines). The mixture was then processed by high-shear 339 homogenization to further decrease particle size. Using an M-110P microfluidizer (Microfluidics), the 340 colloid mixture was processed at 30,000 psi for eleven discrete microfluidization passes. The NLC 341 product was terminally filtered with a 0.22µm polyethersulfone filter and stored at 2°C-8°C until use. Millenium RNA marker (ThermoFisher) was included on each gel with markers at 0.5, 1, 1.5, 2, 2.5, 3, 397 4, 5, 6, and 9 kilobases. Gels were imaged using ethidium bromide protocol on a ChemiDoc MP 398 imaging system (BioRad). Densitometry analysis of the gel images was performed using Image Lab 399 software version 6.1.0 and comparing sample RNA band intensity to the band intensity of an RNA 400 loading control or a freshly complexed (i.e., not stored) control as appropriate for each experiment. 

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An overview of liposome lyophilization and its 606 future potential

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RNA vaccines face challenges during storage and distribution due to their need for frozen storage. In this work, the authors utilize a nanostructured lipid carrier (NLC) delivery system for RNA that can be lyophilized in order to achieve long term storage stability at refrigerated or room temperatures