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ArticleMay 2, 2019

The Skin You Are In: Design-of-Experiments Optimization of Lipid Nanoparticle Self-Amplifying RNA Formulations in Human Skin Explants
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Anna K. Blakney
Anna K. Blakney
Department of Medicine, Imperial College London, London, W21PG, United Kingdom
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http://orcid.org/0000-0002-5812-9689
Paul F. McKay
Paul F. McKay
Department of Medicine, Imperial College London, London, W21PG, United Kingdom
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Bárbara Ibarzo Yus
Bárbara Ibarzo Yus
Department of Medicine, Imperial College London, London, W21PG, United Kingdom
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Judith E. Hunter
Judith E. Hunter
Department of Plastic Surgery, Imperial NHS Trust, London, W68RF, United Kingdom
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Elizabeth A. Dex
Elizabeth A. Dex
Department of Plastic Surgery, Imperial NHS Trust, London, W68RF, United Kingdom
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Robin J. Shattock*
Robin J. Shattock
Department of Medicine, Imperial College London, London, W21PG, United Kingdom
*E-mail: [email protected]
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ACS Nano

Cite this: ACS Nano 2019, 13, 5, 5920–5930

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https://doi.org/10.1021/acsnano.9b01774

Published May 2, 2019

Abstract

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Messenger RNA (mRNA) is a promising tool for biotherapeutics, and self-amplifying mRNA (saRNA) is particularly advantageous, because it results in abundant protein expression and production is easily scalable. While mRNA therapeutics have been shown to be highly effective in small animals, the outcomes do not scale linearly when these formulations are translated to dose-escalation studies in humans. Here, we utilize a design of experiments (DoE) approach to optimize the formulation of saRNA lipid nanoparticles in human skin explants. We first observed that luciferase expression from saRNA peaked after 11 days in human skin. Using DoE inputs of complexing lipid identity, lipid nanoparticle dose, lipid concentration, particle concentration, and ratio of zwitterionic to cationic lipids, we optimized the saRNA-induced luciferase expression in skin explants. Lipid identity and lipid concentration were found to be significant parameters in the DoE model, and the optimized formulation resulted in ∼7-fold increase in luciferase expression, relative to initial 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) formulation. Using flow cytometry, we observed that optimized formulations delivered the saRNA to ∼2% of the resident cells in the human skin explants. Although immune cells comprise only 7% of the total population of cells in skin, immune cells were found to express ∼50% of the RNA. This study demonstrates the powerful combination of using a DoE approach paired with clinically relevant human skin explants to optimize nucleic acid formulations. We expect that this system will be useful for optimizing both formulation and molecular designs of clinically translational nucleic acid vaccines and therapeutics.

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Messenger RNA (mRNA) has emerged as a versatile and advantageous tool for both vaccine and protein replacement therapeutics. RNA has several beneficial features over DNA and protein therapeutics; mRNA is noninfectious and nonintegrating, and the degradation of mRNA by normal cellular processes can be modulated through modifications and delivery vehicles. (1−4) mRNA is the minimal genetic vector, and, thus, antivector immunity is avoided, even after repeated administration. Furthermore, mRNA is known to have adjuvanting properties induced by activation of innate sensing mechanisms, such as toll-like receptors (TLRs) and cytosolic pattern recognition receptors. (5,6) Recently, self-amplifying RNA (saRNA) has been investigated as the next-generation approach for mRNA therapeutics. saRNA vectors are derived from the alphavirus genome (7) and self-replicate upon delivery into cytoplasm, resulting in abundant protein expression and thus minimizing the required doses of RNA. (8−10)

Because mRNA therapeutics are negatively charged and not readily taken up into cells, they must be formulated with a delivery vehicle in order to enable efficient cellular uptake and expression. Previous formulations have included liposomes, (11,12) polyplexes, (13,14) and emulsions, (15,16) typically with a cationic lipid or polymer used to complex/condense the RNA. Preclinical formulations are generally optimized in vitro or in vivo in small animal models, such as mice or rats, in order to assess the effectiveness prior to human clinical trials. Kauffman et al. used an innovative in vivo design of experiments (DoE) approach to optimize the expression of liposome-formulated erythropoietin (EPO)-encoding mRNA, which showed 7-fold greater protein expression. (17) However, when the same formulation was used to deliver siRNA, no enhancement was observed, emphasizing the importance of optimizing each formulation based on the platform and indication.

While mRNA therapeutics have shown promising results in vivo in many small animal models, the transition to humans is poor. For example, Bahl et al. observed remarkable hemagglutinin (HA) inhibition titers in mice (100–1000), ferrets (∼10 000), and nonhuman primates (∼10 000) for HA proteins H10N8- and H7N9-encoding mRNA formulated in lipid nanoparticles (LNPs). (18) However, in the corresponding first-in-human, escalating-dose phase I clinical trial, the HAI titer was merely <100. The reason for this inconsistency is unclear; perhaps it is because both the molecular and formulation components of mRNA therapeutics are optimized in models that are somewhat irrelevant to humans. While small animal models will likely always have a role in preclinical studies, we hypothesize that inherent differences in human innate sensing and tissue architecture pose a barrier to the translation of RNA therapeutics from the laboratory to the clinic. Because of these potential differences between humans and small animal models, we sought to optimize the formulated delivery of saRNA in a relevant human tissue. van den Berg et al. previously optimized a tattooed DNA vaccine in human skin explants; (19) however, to our knowledge, this approach has not been previously applied to optimization of mRNA formulations.

Here, we present the optimization of LNP formulations of saRNA in human skin explants, using a DoE approach to maximize protein expression. We first characterized the temporal kinetics of firefly luciferase (fLuc) in human skin explants. Four complexing lipids were chosen, because of their previous use in nucleic acid formulations, including C12–200 (ionizable), dimethyldioctadecylammonium bromide [(DDA), cationic], 1,2-dioleoyl-3-trimethylammonium-propane [(DOTAP), cationic] and cephalin (zwitterionic). We used input parameters of complexing lipid, LNP dose, lipid concentration, particle concentration, and ratio of cationic to zwitterionic lipid, and a response variable of luciferase expression in human skin explants. Upon completion of the DoE, we used flow cytometry to confirm whether the LNP formulations enhanced saRNA delivery to the cells using GFP expression as a proxy. Finally, we characterized which resident cell types of the human skin explants were taking up and expressing saRNA.

Results and Discussion

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A depiction of the lipid nanoparticle composition and complexing lipids is shown in Scheme 1. saRNA was adsorbed to the outside of LNPs, using a variety of complexing lipids, including ionizable (C12–200), cationic (DDA, DOTAP), and zwitterionic (cephalin) lipids. We used a DoE approach to optimize the luciferase-encoding saRNA delivery into human skin explants by varying different aspects of the formulation, including the complexing lipid, lipid concentration, particle concentration, and the ratio of zwitterionic to cationic lipid. We then assessed whether the enhanced luciferase expression was due to the quantity of cellular uptake, or enhanced expression in individual cells, and then characterized the identity of the skin cells that were expressing saRNA.

Scheme 1. Schematic of Lipid Nanoparticle (LNP) Formulations Used for DoE Analysis: (a) Lipid Nanoparticles Containing a Complexing Lipid, DOPE, and Cholesterol; and (b) Complexing Lipids Used in the DoE Library

saRNA Luciferase Expression Kinetics in Human Skin Explants

While the kinetic profile of saRNA luciferase expression in mice has been well-characterized, (9) we first sought to determine the peak expression of saRNA in human skin explants. A single tissue explant was treated with three separate injections, given simultaneously, containing a dose of 10 μg of fLuc saRNA complexed with DOTAP LNPs at a ratio of total lipid to RNA of 4:1 (w/w) and imaged over the course of 21 days (Figure 1). Quantification of the luciferase activity reveals that a signal is visible after only 24 h (∼40 000 p/s), peaks at day 11 (∼235 000 p/s), but persists for at least 21 days (Figure 1b). Tissue culture and imaging was discontinued after 21 days, because of a visible decline in tissue viability (see Figure S3 in the Supporting Information). As evidenced in Figure 1a, the bleb caused by intradermal (ID) injection of the formulation was not confined to the initial injection space, but rather spreads out across the tissue, which had a volume of ∼3 cm². Thus, for future explant experiments, the tissue was cut into smaller volumes (1 cm²) in order to maintain the intended number of replicates. Furthermore, luciferase quantification for the DoE samples was performed only at 10 days, because this timing was determined to be best-suited to distinguish differences in formulation delivery. van den Berg et al. found that luciferase expression peaked in human skin 24 h after delivery via DNA tattooing, and was depleted by 72 h, (19) likely because of high turnover of the epidermis or gene silencing. (20) We hypothesized that the smaller volume of our tissue explants improved the viability, and the amplifying nature of saRNA yielded a delayed maximum signal. The observed kinetics of saRNA-induced fLuc expression in human skin is similar to the profile observed after intramuscular (IM) injection in mice, wherein the peak expression occurs between 7 and 14 days. (9) The prolonged viability of human skin explants in these experiments enabled us to optimize LNP formulations for saRNA delivery.

Figure 1. Firefly luciferase expression in human skin explants over the course of 21 days after ID injection of three separate, simultaneous injections of 10 μg of saRNA with a mass ratio of lipid to RNA of 4:1 (w/w): (a) time course of ex vivo imaging of luciferase expressed by replicon RNA delivered with the initial formulation of DOTAP LNPs; and (b) quantification of luciferase expression, expressed as the mean total flux (p/s) ± standard deviation for n = 3.

Effects of Complexing Lipid Identity and Lipid-to-RNA Ratio on Luciferase Expression

We first optimized the complexing lipid identity and the ratio of total lipid to RNA on saRNA-induced luciferase expression in human skin explants. We chose a range of complexing lipids to include in the DoE, since they had previously been used in liposomal nucleic acid formulations, including C12–200, (17,21) cephalin, (22) DDA, (23,24) and DOTAP. (25) As shown in Figure 2a, cephalin LNPs were found to have the highest luciferase expression (∼40 000 p/s). C12–200, DDA and DOTAP LNPs had peak luciferase expression of ∼20 000 p/s (Figure 2b). These experiments yielded lower luciferase expression than those presented in Figure 1, because of a lower dose of saRNA and decreased surface area of the skin explant utilized for the DoE. Interestingly, C12–200, cephalin, and DOTAP LNPs were found to have increasing luciferase expression with increasing ratio of lipids to RNA, but DDA had similar luciferase expression levels for all three tested ratios (1:1, 4:1, 18:1 (w/w)). We hypothesize that this occurs because DDA complexes the saRNA more efficiently than the other lipids, and thus the DDA LNPs were not saturated with saRNA at a ratio of 18:1 (w/w). C12–200 and DOTAP have previously been used in siRNA (21,26) and mRNA (17,22,27) delivery, but these lipids have never been formulated in similar LNPs and systematically compared. While both DOTAP and DDA have quaternary, cationic amines, DDA has only two methyl groups and DOTAP has three, which potentially accounts for the more-efficient complexation of DDA LNPs to the saRNA. Zwitterionic lipids such as cephalin are typically used as helper lipids to stabilize liposomes, (28,29) in addition to a cationic lipid that complexes the nucleic acid; however, it has been previously observed that increasing the helper lipid composition of the liposome enhances liposome fusion efficiency. (30) We were interested to evaluate the role of cephalin as a complexing lipid, because it represents a very different class of molecule with a different headgroup and tail saturation. We hypothesize that the primary amine on cephalin is able to directly complex the saRNA, and the comparatively higher molar composition of the cephalin LNPs enhance the fusion of these particles to the human skin cells, resulting in higher luciferase expression. Particle size and charge was not included as an input into the DoE, since this was variable between the formulations (Figure S1 in the Supporting Information); however, the impact of these characteristics on protein expression warrants future studies. Based on these observations, we used a ratio of total lipid to RNA of 18:1 (w/w) for further experimentation and completion of the DoE.

Figure 2. Firefly luciferase expression in human skin explants injected intradermally with LNP formulations with varying lipid identity and LNP dose containing 2 μg of saRNA with medium particle concentration (10⁸ particles/mL) at varying ratios of lipid to RNA (w/w): (a) ex vivo imaging of explants after 11 days and (b) quantification of luciferase imagine expressed as the mean total flux (p/s) ± standard deviation for n = 5.

Effects of Lipid and Particle Concentration on Luciferase Expression

Because we observed that increasing the total ratio of lipids to RNA generally increased the luciferase expression in human skin explants, we then sought to determine whether increasing either the particle or lipid concentration would further enhance luciferase expression. We prepared batches of LNPs with high lipid and particle concentration (H[L]/H[P]) or high lipid and low particle concentration (H[L]/L[P]) for each of the complexing lipids. The high and low particle concentrations were defined as 10⁹ and 10⁷ particles/mL, respectively, while the high lipid concentration was defined as 7.5 mg/mL. Using a ratio of total lipids to RNA of 90:1 (w/w), human skin explants were injected ID and quantified for luciferase expression (Figure 3a). We observed that, for cephalin, DDA, and DOTAP LNPs, the H[L]/H[P] formulation had enhanced luciferase expression, compared to the H[L]/L[P] formulation (Figure 3b), although there was no difference in the C12–200 formulations. The H[L]/H[P] cephalin LNPs had the highest luciferase expression (∼25 000 p/s), followed by the cationic H[L]/H[P] DDA and DOTAP LNPs (∼20 000 p/s) and finally the ionizable C12–200 LNPs (∼10 000 p/s). Despite this trend, the luciferase expression of the cephalin LNPs with low lipid concentration and medium particle concentration (Figure 2) was greater than any of the H[L]/H[P] or H[L]/L[P] formulations, emphasizing that, for the formulations of LNPs, a lower ratio of lipid to RNA is more optimal. We observed slight aggregation of H[L]/H[P] LNPs upon addition of saRNA, and we postulate that this phenomenon is due to exceeding the critical concentration of RNA in the particles, wherein the abrupt addition of RNA to a more highly concentrated particle environment causes aggregation. This observation is similar to previous formulations with polyplexes and saRNA, wherein increasing the ratio of saRNA to cationic polymer results in an aggregation of particles, but this has not been previously reported for LNP formulations of saRNA. (31) These experiments confirm the optimized LNP formulation to be low lipid and medium particle concentrations.

Figure 3. Firefly luciferase expression in human skin explants injected intradermally with LNP formulations with varying lipid and particle concentrations containing 2 μg of saRNA with a ratio of total lipid to RNA of 90:1 (w/w): (a) ex vivo imaging of explants after 11 days and (b) quantification of luciferase imagine expressed as the mean total flux (p/s) ± standard deviation for n = 5. High and low particle concentrations are defined as 10⁹ and 10⁷ particles/mL, respectively, while high lipid concentration is defined as 7.5 mg/mL. H[L]/H[P] denotes high lipid concentration and high particle concentration; H[L]/L[P] denotes high lipid concentration and low particle concentration.

Effects of Combining Zwitterionic and Cationic Complexing Lipids on Luciferase Expression

After observing the higher luciferase expression from LNPs with a single cationic or zwitterionic complexing lipid (Figure 2), we prepared formulations of combinations of cationic and zwitterionic lipids with varying ratios from 10:1 to 0.1:1 while maintaining the low lipid concentration and medium particle concentration. We observed that, generally, the DDA/cephalin LNPs had higher luciferase expression (∼25 000 p/s) than the DOTAP/cephalin LNPs (∼10 000 p/s) (Figure 4). These trends are similar to the formulations, wherein only a single complexing lipid was included (Figure 2). There was no added benefit to combining cationic and zwitterionic lipids into a single LNP. In addition, even the LNP formulations that were primarily cephalin (0.1:1) had lower luciferase expression than the cephalin alone, indicating that combining cationic and zwitterionic lipids is detrimental for saRNA delivery. We postulate that this could be because including cationic lipids alters the net charge of the particles, as shown in Figure S1, thus limiting the cephalin-aided fusion of the particles into cells, reducing uptake. However, this hypothesis warrants further studies to confirm whether this is the case, or if incorporating the cationic lipid complexes irreversibly to the saRNA, rendering it functionally useless within the cytoplasm.

Figure 4. Firefly luciferase expression in human skin explants injected intradermally with LNP formulations with varying ratios of cationic and zwitterionic lipids containing 2 μg of saRNA with a total lipid to RNA ratio of 18:1 (w/w) and medium particle concentration (10⁸ particles/mL): (a) ex vivo imaging of explants after 11 days and (b) quantification of luciferase imagine expressed as mean total flux (p/s) ± standard deviation for n = 5.

Design of Experiments (DoE) Analysis

The DoE approach allows for exploration and characterization of the three-dimensional formulation space in order to identify optimal parameters for saRNA formulation. This approach paired with human skin explants is a clinically relevant way to optimize formulations, as tissue is readily available to facilitate the large number of samples required for an exhaustive full factorial DoE, and ID injections are a clinically viable route of administration for RNA vaccines. (32,33) Using input parameters of complexing lipid identity (C12–200, cephalin, DDA, DOTAP), ratio of total lipid to RNA, lipid concentration, particle concentration, and ratio of cationic to zwitterionic lipid and luciferase expression as the response, we used standard least-squares effect screening to construct a model of which input parameters significantly impact luciferase expression in human skin explants (Figure 5). Our model had a correlation coefficient (R²) of 0.55, with p = 0.0043 for the experimental versus predicted luciferase expression. We found that lipid identity and lipid concentration were the only significant factors. In particular, cephalin lipid identity had the strongest effect, with p = 0.0018. To further visualize the design space, we plotted the fold change of luciferase expression when compared to the original DOTAP LNPs administered at a total lipid-to-RNA ratio of 1:1 (w/w) (see Figure 6). A value of 1 indicates no enhancement of luciferase expression. Cephalin LNPs with a ratio of total lipids to RNA of 18:1 (w/w), low lipid concentration, and medium particle concentration yielded a 7-fold increase in luciferase expression over the original formulation. This observation emphasizes the importance of optimizing the formulation and evidence the practicality of a DoE approach. Previous approaches for DoE optimization of liposomal-formulated mRNA delivery found that the ratio of ionizable lipid to mRNA and the identity of the phospholipid were significant factors in enhancing delivery to the liver. (17) They postulated that increasing the surface charge of the particles enhanced interaction with the cell membrane and resulted in increased particulate uptake. (34) While the ratio of complexing lipid to saRNA was not statistically significant in this model, this is likely due to the DDA LNPs having similar luciferase expression at the tested doses. Cephalin is known to associate with lipid bilayers and cell membranes (35) and, thus, may lightly complex to saRNA and facilitate membrane fusion of the LNPs. We showed that luciferase expression in human skin explants is influenced by the complexing lipid identity and the lipid concentration, and the DoE optimization performed in these experiments resulted in a 7-fold increase in protein expression.

Figure 5. Standard least-squares effect screening DoE analysis of lipid nanoparticle formulation in human skin explants.

Figure 6. Comparison of fold change luciferase expression of tested LNP formulations normalized to original DOTAP formulation (blue bar). Values are expressed fold change total flux (p/s) ± standard deviation.

LNP Delivery and Expression of eGFP saRNA in Human Skin Cells

In order to confirm that our LNP formulations were enhancing the delivery of saRNA into human skin cells within the explant, we used saRNA encoding eGFP and flow cytometry to further investigate these observations. Our main question was whether the LNP formulations were increasing the total number of cells that the saRNA was being expressed in, or whether they were simply facilitating a higher copy number of saRNA per cell, which would result in increased eGFP intensity signal per cell. Based on the results from the DoE, we used each of the LNP formulations with individual complexing lipids, as well as the cephalin H[L]/H[P] and cephalin H[L]/L[P] LNPs in order to account for all the input variables. In keeping with the luciferase expression, we observed that cephalin LNPs resulted in the highest increase in GFP expression (Figure 7a). Furthermore, cephalin LNPs had the highest total number of cells (∼2.5%) expressing GFP (Figure 7b), which was significantly increased above RNA only, which has previously been shown to induce protein expression sans formulation. (36) While the DDA and DOTAP LNPs had higher average percentages of GFP-positive cells, they were not statistically significantly more than RNA only, and there was no increase in the total number of GFP-positive cells with the C12–200, cephalin H[L]/H[P], or cephalin H[L]/L[P] formulations. The RNA only did result in increased GFP intensity above the background (Figure 7a), there is a slight shift in GFP intensity from each of the LNP formulations on a per cell basis, indicating that the LNPs all facilitate increased total amount of saRNA per cell, while the cephalin, DDA, and DOTAP LNPs also increased the total number of positive cells. While the cellular uptake has not been previously characterized for LNP formulations of saRNA, a previous study revealed a similar increase in the number of cells expressing GFP in human skin explants for a pDNA construct transfected into the cells using a tattoo device. (19) It has not yet been defined whether a higher percentage of cells taking up and/or expressing saRNA enhances the immunogenicity of the formulation, and whether there is a balance between RNA expression and innate activation. These results confirm the luciferase DoE results and emphasize the potential for enhanced delivery by increasing the total number of cells affected by saRNA formulations.

Figure 7. GFP expression in human skin cells after intradermal injection with LNP formulations, as determined using flow cytometry: (a) histogram of number of cells expressing GFP for each formulation, and (b) percentage of GFP-positive cells of total live cells for each sample. Bar represents the average ± standard deviation, with a significance of α = 0.05 indicated by an asterisk (*).

Identification of Cells Expressing eGFP in Human Skin Cells

Given the relatively low number of cells observed to be expressing GFP in the human skin explants, we then sought to identify which resident cells are present in the explants and which ones actually express the RNA (see Figure 8, as well as Figure S4 in the Supporting Information). We identified which cells were resident in human skin explants, using a flow cytometry panel capable of identifying epithelial cells (CD45-), fibroblasts (CD90+), NK cells (CD56+), leukocytes (CD45+), Langerhans cells (CD1a+), monocytes (CD14+), dendritic cells (CD11c+), and T cells (CD3+). We found that all of these cells were resident in human skin explants (Figure 8a), and that the majority of cells (93%) are epithelial cells (82.6%) and fibroblasts (10.4%). The immune cells comprised only 7% of the total skin population, which is composed of 0.45% NK cells, 1.33% leukocytes, 3.78% Langerhans cells, 0.24% monocytes, 0.79% dendritic cells, and 0.41% T cells, assuming that the flow cytometry preparation did not bias the presence of any cell types. Interestingly, however, the immune cells comprise a low percentage of the total resident skin cells; they were found be ∼50% of the GFP-expressing cell population (Figure 8b). This observation was true for both formulated and unformulated RNA, with the order of expression as follows: epithelial cells, dendritic cells, Langerhans cells, monocytes, leukocytes, fibroblasts, T cells, and NK cells. We did not observe differences in the cell-viability-tested LNP formulations (see Figure S5 in the Supporting Information). These results indicate that the resident skin immune cells preferentially take up and/or express saRNA, despite formulation. These findings agree strongly with a previous study, which observed that monocytes and dendritic cells were the main cell types that express mRNA after intramuscular or ID injection in rhesus macaques. (37) While the results are consistent, future work is warranted to investigate whether uptake and expression into the plethora of epithelial cells and fibroblasts present in the skin can be enhanced by tailoring the formulation, and whether this would lead to enhanced overall protein expression. Furthermore, it remains to be defined whether preferential uptake by immune cells enhances the immunogenicity of saRNA vaccines and therapeutics, and is likely dependent on the indication of the injection. For example, it may be more beneficial to target immune cells in the context of saRNA vaccines for the prevention of infectious diseases, as opposed to protein replacement therapies.

Figure 8. Identity of cells present in human skin explants and GFP+ cells after ID injection of LNP formulations, as determined by flow cytometry: (a) identity of cells in the population of cells extracted from human skin explants, and (b) identity of GFP-expressing skin cells from explants treated with LNP-formulated RNA. The blue sections of each of the small pie charts indicate the total percentage of immune cells in the GFP+ cell population.

Conclusions

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Here, we optimize LNP formulations of saRNA in human skin explants using a DoE approach. Skin explants were cultured for up to 3 weeks and showed luciferase expression after 24 h, which peaked at 10 days. Of the tested input parameters of lipid identity, including cationic (DDA, DOTAP), ionizable (C12–200), and zwitterionic (cephalin) lipids, the ratio of total lipid to RNA, lipid concentration, particle concentration, and ratio of cationic to zwitterionic lipid, only the lipid identity and lipid concentration significantly affected the saRNA-induced luciferase expression in human skin. Despite general use as a “helper lipid” in LNP formulations, cephalin was found to be the most effective complexing lipid. The DoE enabled a 7-fold increase in luciferase expression, compared to the original formulation. Flow cytometry revealed that all of the formulations enhanced the eGFP expression in human skin cells and paralleled the enhanced delivery with cephalin, DDA, and DOTAP LNPs observed with luciferase imaging studies. Finally, while epithelial cells and fibroblasts were found to comprise the majority of the resident skin cell population, the immune cells were found to express more of the administered RNA, with respect to their proportion of the total cell population. This study demonstrates the powerful combination of using a DoE approach paired with clinically relevant human skin explants to optimize nucleic acid formulations. We expect that this system will be useful for optimizing both formulation and molecular designs of clinically translational nucleic acid vaccines and therapeutics.

Methods

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RNA Synthesis and Purification

Self-amplifying RNA derived from the Venezuelan Equine Encephalitis Virus (VEEV) encoding either firefly luciferase (fLuc) or enhanced green fluorescent protein (eGFP) was prepared using in vitro transcription. pDNA was transformed in Escherichia coli and cultured in 50 mL of LB with 1 mg/mL carbenicillin (Sigma–Aldrich, U.K.) and isolated using a Plasmid Plus Maxiprep kit (QIAGEN, U.K.). pDNA concentration and purity was measured on a NanoDrop One (ThermoFisher, U.K.) and then linearized using MluI for 2 h at 37 °C and heat inactivated at 80 °C for 20 min. Uncapped in vitro RNA transcripts were synthesized using 1 μg of linearized DNA template in a MEGAScript reaction (Promega, U.K.), 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 once with 70% EtOH, centrifuged at 14 000 rpm for 5 min, and then resuspended in UltraPure H₂O. Purified transcripts were then capped using the ScriptCap m⁷G Capping System (CellScript, Madison, WI, USA) and ScriptCap 2′-O-Methyltransferase Kit (CellScript, Madison, WI, USA) simultaneously, according to the manufacturer’s protocol. Capped transcripts were then purified again by LiCl precipitation, resuspended in ultraPure H₂O, and stored at −80 °C until use.

Production of Lipid Nanoparticles

Dimethyldioctadecylammonium bromide (DDA) (Sigma, U.K.), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (Avanti Polar Lipids, Alabaster, AL, USA), and cephalin (soy phosphatidylethanolamine) (Avanti Polar Lipids, Alabaster, AL, USA) were used as received. C12–200 was synthesized by reacting 1 mol equiv of N¹-(2-(4-(2-aminoethyl)piperazin-1-yl)ethyl)ethane-1,2-diamine (Enamine Ltd., Kyiv, Ukraine) with 7 mol equiv of 1,2-epoxydodecane (Sigma, U.K.) at 80 °C for 2.5 days, according to previous protocols. (21) LNPs were prepared on a μEncapsulator 1 System (Dolomite Bio, Royston, U.K.). The lipid solution was prepared by dissolving lipids in 90% EtOH at a total concentration of 1.5 mg/mL, consisting of 35 mol % complexing lipid (C12–200, cephalin, DDA, or DOTAP), 49 mol % cholesterol (Sigma, U.K.) and 16 mol % 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (Avanti Polar Lipids, Alabaster, AL, USA). For high lipid concentration particles, the total lipid concentration was increased to 7.5 mg/mL. One hundred microliters (100 μL) of the lipid was loaded into one side of the μEncapsulator reservoir, while the other side was loaded with 100 μL of citrate buffer (pH 3), and the solutions were then loaded into the corresponding pumps. A 50 μm fluorophilic chip with a T-junction and subsequent PBS dilution channel was used. LNPs were prepared using the following conditions: chip temperature, 70 °C; lipid solution pump pressure, 2000 Pa; citrate buffer pump pressure, 666 Pa; and PBS pump pressure, 2000 Pa. LNPs were purified by dialyzing against PBS in a 3500 MWCO dialysis cartridge (Thermo Fisher, U.K.) overnight. In these studies, high, medium, and low particle concentrations correspond to 10⁹, 10⁸, and 10⁷ particles/mL, respectively, diluted in PBS. For combinations of cationic and zwitterionic lipids, the lipid solutions were prepared such that the total complexing lipid mole percentage was maintained at 35 mol % by varying the ratio of DDA/DOTAP to cephalin (10:1, 1:1, or 0.1:1).

Particle Characterization

LNPs were characterized for size, particle concentration, and surface charge prior to complexation with RNA (Figure S1). One hundred microliters (100 μL) of LNPs was diluted into 900 μL PBS (Sigma, U.K.) and equilibrated at room temperature prior to analysis. The particle size and concentration were characterized on a NanoSight LM10 (Malvern Instruments, U.K.) with NanoSight NTA 3.0 software (Malvern Instruments, U.K.) using an infusion rate of 20, a capture duration of 1 min, a gain of 2, and a camera level of 7. Processing parameters were kept constant for all samples. The surface charge of the LNPs was characterized on a Zetasizer Nano ZS (Malvern Instruments, U.K.) with Zetasizer 7.1 software (Malvern, U.K.) using 850 μL of diluted particles in a 1 mL cuvette and the following settings: material refractive index, 1.529; absorbance, 0.010; dispersant viscosity, 0.8820 cP; refractive index, 1.330; and dielectric constant, 79. Each sample was analyzed for up to 100 runs until the measurement stabilized.

Human Skin Explant Injection, Culture, and Imaging

Surgically resected specimens of human skin tissue were collected at Charing Cross Hospital, Imperial College London, U.K. All tissues were collected after receiving signed informed consent from all 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 its arrival in the laboratory, where it was cut into 1 cm² section, and the subcutaneous layer of fat removed. Explants were incubated at 37 °C with 5% CO₂ in Petri dishes with 10 mL of Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS, 5 mg/mL l-glutamine, and 5 mg/mL penicillin/streptomycin (Thermo Fisher, U.K.). Media was replaced every 3 days, and explants were cultured for up to 21 days. Explants were injected intradermally using a Micro-Fine Demi 0.3 mL syringe (Becton Dickinson, U.K.) with 2 μg of RNA and 25 μL of LNPs in PBS, unless otherwise indicated. After 10 days, explants were inverted such that the epidermis was submerged in the media, and the media was supplemented with 30 ug/mL XenoLight RediJect D-Luciferin (PerkinElmer, U.K.). Samples were imaged with a In Vivo Imaging System (IVIS) FX Pro (Kodak Co., Rochester, NY, USA) equipped with Molecular Imaging Software Version 5.0 (Carestream Health, Rochester, NY, USA), for 60 min. Signal from each tissue explant was analyzed using Molecular Imaging software and expressed as total flux (p/s).

Flow Cytometry

For flow cytometry experiments, eGFP signal was analyzed after 3 days of culture. Skin was minced well with scissors and incubated in 3 mL DMEM supplemented with 1 mg/mL collagenase P (Sigma, U.K.) and 5 mg/mL Dispase II (Sigma, U.K.) for 4 h at 37 °C on a rotational shaker. Digests were then filtered through a 70 μm cell strainer and centrifuged at 1750 rpm for 5 min. Cells were then resuspended in 1 mL of FACS buffer (PBS + 2.5% fetal calf serum (FCS)) at a concentration of 10⁷ cells/mL. One hundred microliters (100 μL) of cell suspension was added to a FACS tube and stained with fixable aqua live/dead cell stain (Thermo Fisher, U.K.) diluted 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 antibodies to identify each cell type, as described in Table S1 in the Supporting Information, for 30 min. Cells were then washed with 2.5 mL of FACS buffer, centrifuged at 1750 rpm for 5 min, and resuspended in 250 μL of PBS. Cells were fixed with 250 μL of 3% paraformaldehyde for a total final concentration of 1.5% and refrigerated until flow cytometry analysis. Samples were analyzed on a LSRFortessa (BD Biosciences, U.K.) with FACSDiva software (BD Biosciences, U.K.) with 30 000 acquired events. Gating strategy is shown in Figure S2 in the Supporting Information. GFP positive cells were quantified using FlowJo Version 10 (FlowJo LLC, Ashland, OR, USA).

Design of Experiment and Statistical Analysis

DoE analysis was performed in JMP, version 13.0, using a full factorial design with complexing lipid identity (C12–200, cephalin, DDA, DOTAP), lipid concentration (high, low), particle concentration (high, low), and ratio of cationic lipid to zwitterionic lipid (10:1, 1:1, 0.1:1) as input factors (Table 1), and luciferase expression in human skin explants after 10 days as the response. The data were analyzed using a fit model of standard least-squares for effect screening with the model effects designated as first- and second-order effects only. Nonsignificant effects were excluded from the model. Graphs were prepared in GraphPad Prism software, version 7.0. Flow cytometry statistical analysis was performed in Prism software, using a two-tailed t test with α = 0.05, which was used to indicate significance.

Table 1. Specifications of Design of Experiment Input Parameters

lipid identity	ratio of total lipid to RNA (w/w)	lipid concentration	particle concentration	ratio cationic to zwitterionic lipids(mol/mol)	ID #
C12–200	18:1	medium	medium	–	1
	4:1	medium	medium	–	2
	1:1	medium	medium	–	3
	90:1	high	high		4
	90:1	high	low		5

cephalin	18:1	medium	medium	–	6
	4:1	medium	medium	–	7
	1:1	medium	medium	–	8
	90:1	high	high		9
	90:1	high	low		10

DDA	18:1	medium	medium	–	11
	4:1	medium	medium	–	12
	1:1	medium	medium	–	13
	18:1	medium	medium	10:1	14
	18:1	medium	medium	1:1	15
				0.1:1	16
	90:1	high	high		17
	90:1	high	low		18

DOTAP	18:1	high/low	high/low	–	19
	4:1	low	low	–	20
	1:1	low	low	–	21
	18:1	low	low	10:1	22
	18:1	low	low	1:1	23
	18:1	low	low	0.1:1	24
	90:1	high	high		25
	90:1	high	low		26

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.9b01774.

LNP characterization of particle size and surface charge (Figure S1); eGFP expression gating strategy (Figure S2); skin explant cell viability (Figure S3); identity of cells present in total population and GFP+ cells (Figure S4); cell viability of human skin explants after LNP-formulation saRNA injection (Figure S5); antibodies used for flow cytometry (Table S1) (PDF)

nn9b01774_si_001.pdf (459.47 kb)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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Corresponding Author
- Robin J. Shattock - Department of Medicine, Imperial College London, London, W21PG, United Kingdom; Email: [email protected]
Authors
- Anna K. Blakney - Department of Medicine, Imperial College London, London, W21PG, United Kingdom; http://orcid.org/0000-0002-5812-9689
- Paul F. McKay - Department of Medicine, Imperial College London, London, W21PG, United Kingdom
- Bárbara Ibarzo Yus - Department of Medicine, Imperial College London, London, W21PG, United Kingdom
- Judith E. Hunter - Department of Plastic Surgery, Imperial NHS Trust, London, W68RF, United Kingdom
- Elizabeth A. Dex - Department of Plastic Surgery, Imperial NHS Trust, London, W68RF, United Kingdom
Author Contributions
A.K.B., P.F.M., and R.J.S. designed the experiments. A.K.B., B.I.Y., and P.F.M. performed the experiments and analyzed the data. A.K.B., B.I.Y., P.F.M., J.E.H., E.A.D., and R.J.S. wrote the manuscript. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Funding
Funding was provided by a Whitaker Post-Doctoral Fellowship (to A.K.B.), Innovate UK, the EPSRC Future Vaccines Manufacturing Research Hub at Imperial College, and the Imperial Confidence in Concept (ICiC) scheme (No. RSRO P75207).
Notes
The authors declare no competing financial interest.

Acknowledgments

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We gratefully acknowledge the nursing team and patients at Charing Cross Hospital and Dormeur Investment Service Ltd. for providing funds to purchase equipment used in these studies.

References

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McCullough, Kenneth C.; Bassi, Isabelle; Milona, Panagiota; Suter, Rolf; Thomann-Harwood, Lisa; Englezou, Pavlos; Demoulins, Thomas; Ruggli, Nicolas
Molecular Therapy--Nucleic Acids (2014), 3 (7), e173CODEN: MTAOC5; ISSN:2162-2531. (Nature Publishing Group)
Self-amplifying replicon RNA (RepRNA) possesses high potential for increasing antigen load within dendritic cells (DCs). The major aim of the present work was to define how RepRNA delivered by biodegradable, chitosan-based nanoparticulate delivery vehicles (nanogel-alginate (NGA)) interacts with DCs, and whether this could lead to translation of the RepRNA in the DCs. Although studies employed virus replicon particles (VRPs), there are no reports on biodegradable, nanoparticulate vehicle delivery of RepRNA. VRP studies employed cytopathogenic agents, contrary to DC requirements-slow processing and antigen retention. We employed noncytopathogenic RepRNA with NGA, demonstrating for the first time the efficiency of RepRNA assocn. with nanoparticles, NGA delivery to DCs, and RepRNA internalization by DCs. RepRNA accumulated in vesicular structures, with patterns typifying cytosolic release. This promoted RepRNA translation, in vitro and in vivo. Delivery and translation were RepRNA concn.-dependent, occurring in a kinetic manner. Including cationic lipids with chitosan during nanoparticle formation enhanced delivery and translation kinetics, but was not required for translation of immunogenic levels in vivo. This work describes for the first time the characteristics assocd. with chitosan-nanoparticle delivery of self-amplifying RepRNA to DCs, leading to translation of encoded foreign genes, namely influenza virus hemagglutinin and nucleoprotein.
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Hekele, A.; Bertholet, S.; Archer, J.; Gibson, D. G.; Palladino, G.; Brito, L. A.; Otten, G. R.; Brazzoli, M.; Buccato, S.; Bonci, A.; Casini, D.; Maione, D.; Qi, Z.-Q.; Gill, J. E.; Caiazza, N. C.; Urano, J.; Hubby, B.; Gao, G. F.; Shu, Y.; De Gregorio, E.; Mandl, C. W.; Mason, P. W.; Settembre, E. C.; Ulmer, J. B.; Craig Venter, J.; Dormitzer, P. R.; Rappuoli, R.; Geall, A. J. Rapidly Produced SAM(®) Vaccine against H7N9 Influenza Is Immunogenic in Mice. Emerging Microbes Infect. 2013, 2, 1– 7, DOI: 10.1038/emi.2013.54
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Brazzoli, M.; Magini, D.; Bonci, A.; Buccato, S.; Giovani, C.; Kratzer, R.; Zurli, V.; Mangiavacchi, S.; Casini, D.; Brito, L. M.; De Gregorio, E.; Mason, P. W.; Ulmer, J. B.; Geall, A. J.; Bertholet, S. Induction of Broad-Based Immunity and Protective Efficacy by Self-Amplifying mRNA Vaccines Encoding Influenza Virus Hemagglutinin. J. Virol. 2016, 90, 332, DOI: 10.1128/JVI.01786-15
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Induction of broad-based immunity and protective efficacy by selfamplifying mRNA vaccines encoding influenza virus hemagglutinin
Brazzoli, Michela; Magini, Diletta; Bonci, Alessandra; Buccato, Scilla; Giovani, Cinzia; Kratzer, Roland; Zurli, Vanessa; Mangiavacchi, Simona; Casini, Daniele; Brito, Luis M.; De Gregorio, Ennio; Mason, Peter W.; Ulmer, Jeffrey B.; Geall, Andrew J.; Bertholet, Sylvie
Journal of Virology (2016), 90 (1), 332-344CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)
Seasonal influenza is a vaccine-preventable disease that remains a major health problem worldwide, esp. in immunocompromised populations. The impact of influenza disease is even greater when strains drift, and influenza pandemics can result when animal-derived influenza virus strains combine with seasonal strains. In this study, we used the SAM technol. and characterized the immunogenicity and efficacy of a self-amplifying mRNA expressing influenza virus hemagglutinin (HA) antigen [SAM(HA)] formulated with a novel oil-in-water cationic nanoemulsion. We demonstrated that SAM(HA) was immunogenic in ferrets and facilitated containment of viral replication in the upper respiratory tract of influenza virus-infected animals. In mice, SAM(HA) induced potent functional neutralizing antibody and cellular immune responses, characterized by HA-specific CD4 T helper 1 and CD8 cytotoxic T cells. Furthermore, mice immunized with SAM(HA) derived from the influenza A virus A/California/7/2009 (H1N1) strain (Cal) were protected from a lethal challenge with the heterologous mouse-adapted A/PR/8/1934 (H1N1) virus strain (PR8). Sera derived from SAM(H1-Cal)-immunized animals were not cross-reactive with the PR8 virus, whereas cross-reactivity was obsd. for HA-specific CD4 and CD8 T cells. Finally, depletion of T cells demonstrated that T-cell responses were essential in mediating heterologous protection. If the SAM vaccine platform proves safe, well tolerated, and effective in humans, the fully synthetic SAM vaccine technol. could provide a rapid response platform to control pandemic influenza.
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Kauffman, K. J.; Dorkin, J. R.; Yang, J. H.; Heartlein, M. W.; DeRosa, F.; Mir, F. F.; Fenton, O. S.; Anderson, D. G. Optimization of Lipid Nanoparticle Formulations for mRNA Delivery In Vivo with Fractional Factorial and Definitive Screening Designs. Nano Lett. 2015, 15, 7300– 7306, DOI: 10.1021/acs.nanolett.5b02497
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Optimization of Lipid Nanoparticle Formulations for mRNA Delivery in Vivo with Fractional Factorial and Definitive Screening Designs
Kauffman, Kevin J.; Dorkin, J. Robert; Yang, Jung H.; Heartlein, Michael W.; De Rosa, Frank; Mir, Faryal F.; Fenton, Owen S.; Anderson, Daniel G.
Nano Letters (2015), 15 (11), 7300-7306CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
Intracellular delivery of mRNA (mRNA) has the potential to induce protein prodn. for many therapeutic applications. Although lipid nanoparticles have shown considerable promise for the delivery of small interfering RNAs (siRNA), their utility as agents for mRNA delivery has only recently been investigated. The most common siRNA formulations contain four components: an amine-contg. lipid or lipid-like material, phospholipid, cholesterol, and lipid-anchored polyethylene glycol, the relative ratios of which can have profound effects on the formulation potency. Here, we develop a generalized strategy to optimize lipid nanoparticle formulations for mRNA delivery to the liver in vivo using Design of Expt. (DOE) methodologies including Definitive Screening and Fractional Factorial Designs. By simultaneously varying lipid ratios and structures, we developed an optimized formulation which increased the potency of erythropoietin-mRNA-loaded C12-200 lipid nanoparticles 7-fold relative to formulations previously used for siRNA delivery. Key features of this optimized formulation were the incorporation of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and increased ionizable lipid:mRNA wt. ratios. Interestingly, the optimized lipid nanoparticle formulation did not improve siRNA delivery, indicating differences in optimized formulation parameter design spaces for siRNA and mRNA. We believe the general method described here can accelerate in vivo screening and optimization of nanoparticle formulations with large multidimensional design spaces.
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Bahl, K.; Senn, J. J.; Yuzhakov, O.; Bulychev, A.; Brito, L. A.; Hassett, K. J.; Laska, M. E.; Smith, M.; Almarsson, Ö.; Thompson, J.; Ribeiro, A.; Watson, M.; Zaks, T.; Ciaramella, G. Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9 Influenza Viruses. Mol. Ther. 2017, 25, 1316– 1327, DOI: 10.1016/j.ymthe.2017.03.035
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Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9 Influenza Viruses
Bahl, Kapil; Senn, Joe J.; Yuzhakov, Olga; Bulychev, Alex; Brito, Luis A.; Hassett, Kimberly J.; Laska, Michael E.; Smith, Mike; Almarsson, Orn; Thompson, James; Ribeiro, Amilcar; Watson, Mike; Zaks, Tal; Ciaramella, Giuseppe
Molecular Therapy (2017), 25 (6), 1316-1327CODEN: MTOHCK; ISSN:1525-0024. (Cell Press)
Recently, the World Health Organization confirmed 120 new human cases of avian H7N9 influenza in China resulting in 37 deaths, highlighting the concern for a potential pandemic and the need for an effective, safe, and high-speed vaccine prodn. platform. Prodn. speed and scale of mRNA-based vaccines make them ideally suited to impede potential pandemic threats. Here we show that lipid nanoparticle (LNP)-formulated, modified mRNA vaccines, encoding hemagglutinin (HA) proteins of H10N8 (A/Jiangxi-Donghu/346/2013) or H7N9 (A/Anhui/1/2013), generated rapid and robust immune responses in mice, ferrets, and nonhuman primates, as measured by hemagglutination inhibition (HAI) and microneutralization (MN) assays. A single dose of H7N9 mRNA protected mice from a lethal challenge and reduced lung viral titers in ferrets. Interim results from a first-in-human, escalating-dose, phase 1 H10N8 study show very high seroconversion rates, demonstrating robust prophylactic immunity in humans. Adverse events (AEs) were mild or moderate with only a few severe and no serious events. These data show that LNP-formulated, modified mRNA vaccines can induce protective immunogenicity with acceptable tolerability profiles.
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van den Berg, J. H.; Nuijen, B.; Beijnen, J. H.; Vincent, A.; van Tinteren, H.; Kluge, J.; Woerdeman, L. A. E.; Hennink, W. E.; Storm, G.; Schumacher, T. N.; Haanen, J. B. A. G. Optimization of Intradermal Vaccination by DNA Tattooing in Human Skin. Hum. Gene Ther. 2009, 20, 181– 189, DOI: 10.1089/hum.2008.073
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Optimization of Intradermal Vaccination by DNA Tattooing in Human Skin
van den Berg, Joost H.; Nuijen, Bastiaan; Beijnen, Jos H.; Vincent, Andrew; van Tinteren, Harm; Kluge, Joern; Woerdeman, Leonie A. E.; Hennink, Wim E.; Storm, Gert; Schumacher, Ton N.; Haanen, John B. A. G.
Human Gene Therapy (2009), 20 (3), 181-189CODEN: HGTHE3; ISSN:1043-0342. (Mary Ann Liebert, Inc.)
The intradermal administration of DNA vaccines by tattooing is a promising delivery technique for genetic immunization, with proven high immunogenicity in mice and in nonhuman primates. However, the parameters that result in optimal expression of DNA vaccines that are applied by this strategy to human skin are currently unknown. To address this issue we set up an ex vivo human skin model in which DNA vaccine-induced expression of reporter proteins could be monitored longitudinally. Using this model we demonstrate the following: First, the vast majority of cells that express DNA vaccine-encoded antigen in human skin are formed by epidermal keratinocytes, with only a small fraction (about 1%) of antigen-pos. epidermal Langerhans cells. Second, using full randomization of DNA tattoo variables we show that an increase in DNA concn., needle depth, and tattoo time all significantly increase antigen expression (p < 0.001), with DNA concn. forming the most crit. variable influencing the level of antigen expression. Finally, in spite of the marked immunogenicity of this vaccination method in animal models, transfection efficiency of the technique is shown to be extremely low, estd. at approx. 2 to 2000 out of 1 × 1010 copies of plasmid applied. This finding, coupled with the obsd. dependency of antigen expression on DNA concn., suggests that the development of strategies that can enhance in vivo transfection efficacy would be highly valuable. Collectively, this study shows that an ex vivo human skin model can be used to det. the factors that control vaccine-induced antigen expression and define the optimal parameters for the evaluation of DNA tattoo or other dermal delivery techniques in phase 1 clin. trials.
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Gelfant, S. Of Mice and Men” the Cell Cycle in Human Epidermis In Vivo. J. Invest. Dermatol. 1982, 78, 296– 299, DOI: 10.1111/1523-1747.ep12507367
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"Of mice and men" the cell cycle in human epidermis in vivo
Gelfant S
The Journal of investigative dermatology (1982), 78 (4), 296-9 ISSN:0022-202X.
This article deals with and compares cell cycle information obtained in mouse and in human epidermis in vivo. In order to compare data in mouse and in man, DNA labeling and mitotic index experiments were performed to obtain cell cycle information in normal human epidermis in vivo. Experiments were also performed on genetically inbred and outbred strains of mice--to provide a clue to the differences observed between mouse and man. The article makes the following points: 1. In contrast to mouse epidermis, there are no consistent diurnal fluctuations in and there is no cell kinetic relationship between mitotic and DNA-labeling indices in normal human epidermis in vivo. The variability from individual to individual in human subjects and the lack of cell cycle-related circadian fluctuations, preclude the use of statistical analysis and the use of conventional cell kinetic principles in understanding epidermal cell proliferation in man and may preclude the use of circadian rhythmicity for therapy scheduling. 2. The consistent and intelligible cell cycle information obtained in laboratory mice (as compared to man) is not due to the genetically inbred condition of mice. 3. This report introduces the use of ambient temperature as a potential nontoxic cell cycle tool for manipulating epidermal cell proliferation in the therapy of human proliferative skin diseases.
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Love, K. T.; Mahon, K. P.; Levins, C. G.; Whitehead, K. A.; Querbes, W.; Dorkin, J. R.; Qin, J.; Cantley, W.; Qin, L. L.; Racie, T.; Frank-Kamenetsky, M.; Yip, K. N.; Alvarez, R.; Sah, D. W. Y.; de Fougerolles, A.; Fitzgerald, K.; Koteliansky, V.; Akinc, A.; Langer, R.; Anderson, D. G. Lipid-Like Materials for Low-Dose, In Vivo Gene Silencing. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 1864– 1869, DOI: 10.1073/pnas.0910603106
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Lipid-like materials for low-dose, in vivo gene silencing
Love, Kevin T.; Mahon, Kerry P.; Levins, Christopher G.; Whitehead, Kathryn A.; Querbes, William; Dorkin, J. Robert; Qin, June; Cantley, William; Qin, Liu Liang; Racie, Timothy; Frank-Kamenetsky, Maria; Yip, Ka Ning; Alvarez, Rene; Sah, Dinah W. Y.; de Fougerolles, Antonin; Fitzgerald, Kevin; Koteliansky, Victor; Akinc, Akin; Langer, Robert; Anderson, Daniel G.
Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (5), 1864-1869, S1864/1-S1864/6CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
Significant effort has been applied to discover and develop vehicles which can guide small interfering RNAs (siRNA) through the many barriers guarding the interior of target cells. While studies have demonstrated the potential of gene silencing in vivo, improvements in delivery efficacy are required to fulfill the broadest potential of RNA interference therapeutics. Through the combinatorial synthesis and screening of a different class of materials, a formulation has been identified that enables siRNA-directed liver gene silencing in mice at doses below 0.01 mg/kg. This formulation was also shown to specifically inhibit expression of five hepatic genes simultaneously, after a single injection. The potential of this formulation was further validated in nonhuman primates, where high levels of knockdown of the clin. relevant gene transthyretin was obsd. at doses as low as 0.03 mg/kg. To our knowledge, this formulation facilitates gene silencing at orders-of-magnitude lower doses than required by any previously described siRNA liver delivery system.
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Ball, R. L.; Hajj, K. A.; Vizelman, J.; Bajaj, P.; Whitehead, K. A. Lipid Nanoparticle Formulations for Enhanced Co-Delivery of siRNA and mRNA. Nano Lett. 2018, 18, 3814– 3822, DOI: 10.1021/acs.nanolett.8b01101
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Lipid nanoparticle formulations for enhanced co-delivery of siRNA and mRNA
Ball, Rebecca L.; Hajj, Khalid A.; Vizelman, Jamie; Bajaj, Palak; Whitehead, Kathryn A.
Nano Letters (2018), 18 (6), 3814-3822CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
Although mRNA and siRNA have significant therapeutic potential, their simultaneous delivery has not been previously explored. To facilitate the treatment of diseases assocd. with aberrant gene upregulation and downregulation, we sought to co-formulate siRNA and mRNA in a single lipidoid nanoparticle (LNP) formulation. We accommodated the distinct mol. characteristics of mRNA and siRNA in a formulation consisting of an ionizable and biodegradable amine-contg. lipidoid, cholesterol, DSPC, DOPE, and PEG-lipid. Surprisingly, the co-formulation of siRNA and mRNA in the same LNP enhanced the efficacy of both drugs in vitro and in vivo. Compared to LNPs encapsulating siRNA only, co-formulated LNPs improved Factor VII gene silencing in mice from 44 to 87% at an siRNA dose of 0.03 mg/kg. Co-formulation also improved mRNA delivery, as a 0.5 mg/kg dose of mRNA co-formulated with siRNA induced three times the luciferase protein expression compared to when siRNA was not included. As not all gene therapy applications require both RNA drugs, we sought to extend the benefit of co-formulated LNPs to formulations encapsulating only a single type of RNA. We accomplished this by substituting the "helper" RNA with a neg. charged polymer, polystyrenesulfonate (PSS). LNPs contg. PSS mediated the same level of protein silencing or expression as std. LNPs using 2-3-fold less RNA. For example, LNPs formulated with and without PSS induced 50% Factor VII silencing at siRNA doses of 0.01 and 0.03 mg/kg, resp. Together, these studies demonstrate potent co-delivery of siRNA and mRNA and show that inclusion of a neg. charged "helper polymer" enhances the efficacy of LNP delivery systems.
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Henriksen-Lacey, M.; Christensen, D.; Bramwell, V. W.; Lindenstrøm, T.; Agger, E. M.; Andersen, P.; Perrie, Y. Comparison of the Depot Effect and Immunogenicity of Liposomes Based on Dimethyldioctadecylammonium (DDA), 3β-[N-(N′,N′-Dimethylaminoethane)Carbomyl] Cholesterol (DC-Chol), and 1,2-Dioleoyl-3-Trimethylammonium Propane (DOTAP): Prolonged Liposome Retention Mediates Stronger Th1 Responses. Mol. Pharmaceutics 2011, 8, 153– 161, DOI: 10.1021/mp100208f
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Comparison of the Depot Effect and Immunogenicity of Liposomes Based on Dimethyldioctadecylammonium (DDA), 3β-[N-(N',N'-Dimethylaminoethane)carbomyl] Cholesterol (DC-Chol), and 1,2-Dioleoyl-3-trimethylammonium Propane (DOTAP): Prolonged Liposome Retention Mediates Stronger Th1 Responses
Henriksen-Lacey, Malou; Christensen, Dennis; Bramwell, Vincent W.; Lindenstrom, Thomas; Agger, Else Marie; Andersen, Peter; Perrie, Yvonne
Molecular Pharmaceutics (2011), 8 (1), 153-161CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)
The immunostimulatory capacities of cationic liposomes are well-documented and are attributed both to inherent immunogenicity of the cationic lipid and more phys. capacities such as the formation of antigen depots and antigen delivery. Very few studies have however been conducted comparing the immunostimulatory capacities of different cationic lipids. In the present study we therefore chose to investigate three of the most well-known cationic liposome-forming lipids as potential adjuvants for protein subunit vaccines. The ability of 3β-[N-(N',N'-dimethylaminoethane)carbomyl] cholesterol (DC-Chol), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), and dimethyldioctadecylammonium (DDA) liposomes incorporating immunomodulating trehalose dibehenate (TDB) to form an antigen depot at the site of injection (SOI) and to induce immunol. recall responses against coadministered tuberculosis vaccine antigen Ag85B-ESAT-6 are reported. Furthermore, phys. characterization of the liposomes is presented. Our results suggest that liposome compn. plays an important role in vaccine retention at the SOI and the ability to enable the immune system to induce a vaccine specific recall response. While all three cationic liposomes facilitated increased antigen presentation by antigen presenting cells, the monocyte infiltration to the SOI and the prodn. of IFN-γ upon antigen recall was markedly higher for DDA and DC-Chol based liposomes which exhibited a longer retention profile at the SOI. A long-term retention and slow release of liposome and vaccine antigen from the injection site hence appears to favor a stronger Th1 immune response.
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De Serrano, L. O.; Burkhart, D. J. Liposomal Vaccine Formulations as Prophylactic Agents: Design Considerations for Modern Vaccines. J. Nanobiotechnol. 2017, 15, 83, DOI: 10.1186/s12951-017-0319-9
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Liposomal vaccine formulations as prophylactic agents: design considerations for modern vaccines
De Serrano, Luis O.; Burkhart, David J.
Journal of Nanobiotechnology (2017), 15 (), 83/1-83/23CODEN: JNOAAO; ISSN:1477-3155. (BioMed Central Ltd.)
A review. Vaccinol. is one of the most important cornerstones in modern medicine, providing better quality of life. The human immune system is composed of innate and adaptive immune processes that interplay when infection occurs. Innate immunity relies on pathogen-assocd. mol. patterns which are recognized by pathogen recognition receptors localized in antigen presenting cells. After antigen processing and presentation, CD4+ T cell polarization occurs, further leading to B cell and CD8+ activation and humoral and cell-mediated adaptive immune responses. Liposomes are being employed as vaccine technologies and their design is of importance to ensure proper immune responses. Physicochem. parameters like liposome size, charge, lamellarity and bilayer fluidity must be completely understood to ensure optimal vaccine stability and efficacy. Liposomal vaccines can be developed to target specific immune cell types for the induction of certain immune responses. In this review, we will present promising liposomal vaccine approaches for the treatment of important viral, bacterial, fungal and parasitic infections (including tuberculosis, TB). Cationic liposomes are the most studied liposome types due to their enhanced interaction with the neg. charged immune cells. Thus, a special section on the cationic lipid dimethyldioctadecylammonium and TB is also presented.
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Martino, S.; di Girolamo, I.; Tiribuzi, R.; D’Angelo, F.; Datti, A.; Orlacchio, A. Efficient siRNA Delivery by the Cationic Liposome DOTAP in Human Hematopoietic Stem Cells Differentiating into Dendritic Cells. J. Biomed. Biotechnol. 2009, 2009, 410260, DOI: 10.1155/2009/410260
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Efficient siRNA delivery by the cationic liposome DOTAP in human hematopoietic stem cells differentiating into dendritic cells
Martino Sabata; di Girolamo Ilaria; Tiribuzi Roberto; D'Angelo Francesco; Datti Alessandro; Orlacchio Aldo
Journal of biomedicine & biotechnology (2009), 2009 (), 410260 ISSN:.
RNA interference technology is an ideal strategy to elucidate the mechanisms associated with human CD34(+) hematopoietic stem cell differentiation into dendritic cells. Simple manipulations in vitro can unequivocally yield alloreactive or tolerogenic populations, suggesting key implications of biochemical players that might emerge as therapeutic targets for cancer or graft-versus-host disease. To knockdown proteins typically involved in the biology of dendritic cells, we employed an siRNA delivery system based on the cationic liposome DOTAP as the carrier. Freshly-isolated CD34(+) cells were transfected with siRNA for cathepsin S with negligible cytotoxicity and transfection rates (>60%) comparable to the efficiency shown by lentiviral vectors. Further, cathepsin S knockdown was performed during both cell commitment and through the entire 14-day differentiation process with repeated transfection rounds that had no effect per se on cell development. Tested in parallel, other commercially-available chemical reagents failed to meet acceptable standards. In addition to safe and practical handling, a direct advantage of DOTAP over viral-mediated techniques is that transient silencing effects can be dynamically appraised through the recovery of targeted proteins. Thus, our findings identify DOTAP as an excellent reagent for gene silencing in resting and differentiating CD34(+) cells, suggesting a potential for applications in related preclinical models.
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Kaneda, M. M.; Sasaki, Y.; Lanza, G. M.; Milbrandt, J.; Wickline, S. A. Mechanisms of Nucleotide Trafficking During siRNA Delivery to Endothelial Cells Using Perfluorocarbon Nanoemulsions. Biomaterials 2010, 31, 3079– 3086, DOI: 10.1016/j.biomaterials.2010.01.006
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Mechanisms of nucleotide trafficking during siRNA delivery to endothelial cells using perfluorocarbon nanoemulsions
Kaneda, Megan M.; Sasaki, Yo; Lanza, Gregory M.; Milbrandt, Jeffrey; Wickline, Samuel A.
Biomaterials (2010), 31 (11), 3079-3086CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
RNA interference (RNAi) is a useful in vitro research tool, but its application as a safe and effective therapeutic agent may benefit from improved understanding of mechanisms of exogenous siRNA delivery, including cell trafficking and sorting patterns. We report the development of a transfection reagent for siRNA delivery which employs a distinctive non-digestive mode of particle-cell membrane interaction through the formation of a hemifusion complex resulting in lipid raft transport of cargo to the cytosol, bypassing the usual endosomal nanoparticle uptake pathway. We further demonstrate markedly enhanced efficacy over conventional transfection agents for suppressing endothelial cell expression of upregulated vascular adhesion mols.
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Zhang, X.; Li, B.; Luo, X.; Zhao, W.; Jiang, J.; Zhang, C.; Gao, M.; Chen, X.; Dong, Y. Biodegradable Amino-Ester Nanomaterials for Cas9 mRNA Delivery In Vitro and In Vivo. ACS Appl. Mater. Interfaces 2017, 9, 25481– 25487, DOI: 10.1021/acsami.7b08163
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Biodegradable Amino-Ester Nanomaterials for Cas9 mRNA Delivery in Vitro and in Vivo
Zhang, Xinfu; Li, Bin; Luo, Xiao; Zhao, Weiyu; Jiang, Justin; Zhang, Chengxiang; Gao, Min; Chen, Xiaofang; Dong, Yizhou
ACS Applied Materials & Interfaces (2017), 9 (30), 25481-25487CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
Efficient and safe delivery of the CRISPR/Cas system is one of the key challenges for genome-editing applications in humans. Herein, we designed and synthesized a series of biodegradable lipidlike compds. contg. ester groups for the delivery of mRNA-encoding Cas9. Two lead materials, termed N-methyl-1,3-propanediamine (MPA)-A and MPA-Ab, showed a tunable rate of biodegrdn. MPA-A with linear ester chains was degraded dramatically faster than MPA-Ab with branched ester chains in the presence of esterase or in wild-type mice. Most importantly, MPA-A and MPA-Ab demonstrated efficient delivery of Cas9 mRNA both in vitro and in vivo. Consequently, these biodegradable lipidlike nanomaterials merit further development as genome-editing delivery tools for biol. and therapeutic applications.
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Kim, B.-K.; Hwang, G.-B.; Seu, Y.-B.; Choi, J.-S.; Jin, K. S.; Doh, K.-O. DOTAP/DOPE Ratio and Cell Type Determine Transfection Efficiency with DOTAP-Liposomes. Biochim. Biophys. Acta, Biomembr. 2015, 1848, 1996– 2001, DOI: 10.1016/j.bbamem.2015.06.020
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DOTAP/DOPE ratio and cell type determine transfection efficiency with DOTAP-liposomes
Kim, Bieong-Kil; Hwang, Guen-Bae; Seu, Young-Bae; Choi, Jong-Soo; Jin, Kyeong Sik; Doh, Kyung-Oh
Biochimica et Biophysica Acta, Biomembranes (2015), 1848 (10_Part_A), 1996-2001CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
The effects of lipid compns. on their physicochem. properties and transfection efficiencies were investigated. Four liposome formulations with different 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) to dioleoylphosphatidylethanolamine (DOPE) wt. ratios were investigated, i.e., wt. ratios 1:0 (T1P0), 3:1 (T3P1), 1:1 (T1P1), and 1:3 (T1P3). Mean sizes of liposomes were influenced by their lipid compn. and the prepn. concn. at the time of sonication. Zeta potentials of liposomes were inversely correlated with their liposome sizes. However, neither liposome sizes nor zeta potentials were correlated with transfection efficiency. The optimum compn. of liposomes was cell-line dependent (T1P0 and T3P1 for Huh7 and AGS, T3P1 and T1P1 for COS7, and T1P1 and T1P3 for A549). The shape of lipoplexes was changed from lamellar to inverted hexagonal structure according to the increased ratio of DOPE, but there was no definite advantage of specific structure in transfection efficiency throughout all used cell lines. However, cellular internalization was consistently faster in T1P0, T3P1, T1P1 compared to T1P3 in all cell lines, suggesting the importance of endosomal escape. Our findings show that the transfection efficiency of DOTAP liposomes is mainly influenced by lipid compn. and cell type, and not by size or zeta potential.
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Mével, M.; Haudebourg, T.; Colombani, T.; Peuziat, P.; Dallet, L.; Chatin, B.; Lambert, O.; Berchel, M.; Montier, T.; Jaffrès, P.-A.; Lehn, P.; Pitard, B. Important Role of Phosphoramido Linkage in Imidazole-Based Dioleyl Helper Lipids for Liposome Stability and Primary Cell Transfection. J. Gene Med. 2016, 18, 3– 15, DOI: 10.1002/jgm.2869
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Important role of phosphoramido linkage in imidazole-based dioleyl helper lipids for liposome stability and primary cell transfection
Mevel, Mathieu; Haudebourg, Thomas; Colombani, Thibault; Peuziat, Pauline; Dallet, Laurence; Chatin, Benoit; Lambert, Olivier; Berchel, Mathieu; Montier, Tristan; Jaffres, Paul-Alain; Lehn, Pierre; Pitard, Bruno
Journal of Gene Medicine (2016), 18 (1-3), 3-15CODEN: JGMEFG; ISSN:1521-2254. (Wiley-Blackwell)
Background : To optimize synthetic gene delivery systems, there is a need to develop more efficient lipid formulations. Most cationic lipid formulations contain 'helper' neutral lipids because of their ability to increase DNA delivery, in particular by improving endosomal escape of DNA mols. via the pH-buffering effect of protonatable groups and/or fusion with the lipid bilayer of endosomes. Methods : We evaluated the influence of the linker structure between the two oleyl chains in the helper lipid on transfection efficiency in cell lines, as well as in primary cells (hepatocytes/cardiomyocytes). We reported the synthesis of two new pH-buffering imidazole helper lipids characterized by a polar headgroup contg. one (compd. 6) or two (compd. 5) imidazole groups and two oleyl chains linked by an amide group. We studied their assocn. with the aminoglycoside lipidic deriv. dioleylsuccinylparomomycin (DOSP), which contains two oleyl chains linked to the aminoglycoside polar headgroup via an amide function. We compared the morphol. and transfection properties of such binary liposomes of DOSP/5 and DOSP/6 with those of liposomes combining DOSP with another imidazole-based dioleyl helper lipid (MM27) in which a phosphoramido group acts as a linker between the two oleyl chains and imidazole function. Results : The phosphoramido linker in the helper lipid induces a major difference in terms of morphol. and resistance to decomplexation at phys. pH for DOSP/helper lipid complexes. Conclusions : This hybrid dioleyl linker compn. of DOSP/MM27 led to higher transfection efficiency in cell lines and in primary cells compared to complexes with homogeneous dioleyl linker. Copyright © 2015 John Wiley & Sons, Ltd.
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Cavalcanti, R. R. M.; Lira, R. B.; Riske, K. A. Study of the Fusion Mechanism of Fusogenic Cationic Liposomes with Anionic Model Membranes. Biophys. J. 2018, 114, 606a, DOI: 10.1016/j.bpj.2017.11.3314
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Self-amplifying replicon RNA (RepRNA) are large mols. (12-14 kb); their self-replication amplifies mRNA template nos., affording several rounds of antigen prodn., effectively increasing vaccine antigen payloads. Their sensitivity to RNase-sensitivity and inefficient uptake by dendritic cells (DCs) - abs. requirements for vaccine design - were tackled by condensing RepRNA into synthetic, nanoparticulate, polyethylenimine (PEI)-polyplex delivery vehicles. Polyplex-delivery formulations for small RNA mols. cannot be transferred to RepRNA due to its greater size and complexity; the N:P charge ratio and impact of RepRNA folding would influence polyplex condensation, post-delivery decompaction and the cytosolic release essential for RepRNA translation. Polyplex-formulations proved successful for delivery of RepRNA encoding influenza virus hemagglutinin and nucleocapsid to DCs. Cytosolic translocation was facilitated, leading to RepRNA translation. This efficacy was confirmed in vivo, inducing both humoral and cellular immune responses. Accordingly, this paper describes the first PEI-polyplexes providing efficient delivery of the complex and large, self-amplifying RepRNA vaccines.
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Pardi, N.; Parkhouse, K.; Kirkpatrick, E.; McMahon, M.; Zost, S. J.; Mui, B. L.; Tam, Y. K.; Karikó, K.; Barbosa, C. J.; Madden, T. D.; Hope, M. J.; Krammer, F.; Hensley, S. E.; Weissman, D. Nucleoside-Modified mRNA Immunization Elicits Influenza Virus Hemagglutinin Stalk-Specific Antibodies. Nat. Commun. 2018, 9, 3361, DOI: 10.1038/s41467-018-05482-0
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Nature communications (2018), 9 (1), 3361 ISSN:.
Currently available influenza virus vaccines have inadequate effectiveness and are reformulated annually due to viral antigenic drift. Thus, development of a vaccine that confers long-term protective immunity against antigenically distant influenza virus strains is urgently needed. The highly conserved influenza virus hemagglutinin (HA) stalk represents one of the potential targets of broadly protective/universal influenza virus vaccines. Here, we evaluate a potent broadly protective influenza virus vaccine candidate that uses nucleoside-modified and purified mRNA encoding full-length influenza virus HA formulated in lipid nanoparticles (LNPs). We demonstrate that immunization with HA mRNA-LNPs induces antibody responses against the HA stalk domain of influenza virus in mice, rabbits, and ferrets. The HA stalk-specific antibody response is associated with protection from homologous, heterologous, and heterosubtypic influenza virus infection in mice.
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MRNA vaccines elicit a potent immune response including antibodies and cytotoxic T cells. MRNA vaccines are currently evaluated in clin. trials for cancer immunotherapy applications, but also have great potential as prophylactic vaccines. Efficient delivery of mRNA vaccines will be key for their success and translation to the clinic. Among potential nonviral vectors, lipid nanoparticles are particularly promising. Indeed, lipid nanoparticles can be synthesized with relative ease in a scalable manner, protect the mRNA against degrdn., facilitate endosomal escape, can be targeted to the desired cell type by surface decoration with ligands, and as needed, can be codelivered with adjuvants.
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A review. An understanding of the interactions between nanoparticles and biol. systems is of significant interest. Studies aimed at correlating the properties of nanomaterials such as size, shape, chem. functionality, surface charge, and compn. with biomol. signaling, biol. kinetics, transportation, and toxicity in both cell culture and animal expts. are under way. These fundamental studies will provide a foundation for engineering the next generation of nanoscale devices. Here, the authors provide rationales for these studies, review the current progress in studies of the interactions of nanomaterials with biol. systems, and provide a perspective on the long-term implications of these findings.
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An Extreme Strategy for the Production of Hybridoma
Golestani, Reza; Pourfathollah, Ali Akbar; Moazzeni, Seyed Mohammad
Hybridoma (2009), 28 (2), 139-144CODEN: HYBRAV; ISSN:1554-0014. (Mary Ann Liebert, Inc.)
The ethical issues surrounding human immunization hamper the prodn. of human monoclonal antibody through scarcity of immunized B cells in peripheral blood. This defect can be compensated in part by improvement of hybridoma prodn. techniques. We have developed a new strategy to bypass the toxic effects of polyethylene glycol (PEG) as fusogenic reagent and hypoxanthine aminoptrin thymidine (HAT) as selective medium on newly fused cells. The Epstein-Barr virus (EBV) transformed peripheral blood mononuclear cells (PBMC) of accidentally Rh antigen sensitized persons were fused using cephalin as fugenic reagent, with emetine and actinomycin D pretreated heteromyeloma cells. Our results showed that 19-34% of EBV-transformed B cells were grown as hybridoma clones following selection. This extreme improvement in hybridoma prodn. rate may end the fusion efficiency problem and make hybridoma prodn. a plug-and-play technol.
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Phua, K. K. L.; Leong, K. W.; Nair, S. K. Transfection Efficiency and Transgene Expression Kinetics of mRNA Delivered in Naked and Nanoparticle Format. J. Controlled Release 2013, 166, 227– 233, DOI: 10.1016/j.jconrel.2012.12.029
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Transfection efficiency and transgene expression kinetics of mRNA delivered in naked and nanoparticle format
Phua, Kyle K. L.; Leong, Kam W.; Nair, Smita K.
Journal of Controlled Release (2013), 166 (3), 227-233CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
Transfection efficiencies and transgene expression kinetics of mRNA, an emerging class of nucleic acid-based therapeutics, were poorly characterized. In this study, the authors evaluated transfection efficiencies of mRNA delivered in naked and nanoparticle format in vitro and in vivo using GFP and luciferase as reporters. While mRNA nanoparticles transfect primary human and mouse dendritic cells (DCs) efficiently in vitro, naked mRNA could not produce any detectable gene product. The protein expression of nanoparticle-mediated transfection in vitro peaks rapidly within 5-7 h and decays in a biphasic manner. In vivo, naked mRNA is more efficient than mRNA nanoparticles when administered s.c. In contrast, mRNA nanoparticle performs better when administered intranasally and i.v. Gene expression is most transient when delivered i.v. in nanoparticle format with an apparent half-life of 1.4 h and lasts less than 24 h, and most sustained when delivered in the naked format s.c. at the base of tail with an apparent half-life of 18 h and persists for at least 6 days. Notably, exponential decreases in protein expression are consistently obsd. post-delivery of mRNA in vivo regardless of the mode of delivery (naked or nanoparticle) or the site of administration. This study elucidates the performance of mRNA transfection and suggests a niche for mRNA therapeutics when predictable in vivo transgene expression kinetics is imperative.
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Liang, F.; Lindgren, G.; Lin, A.; Thompson, E. A.; Ols, S.; Röhss, J.; John, S.; Hassett, K.; Yuzhakov, O.; Bahl, K.; Brito, L. A.; Salter, H.; Ciaramella, G.; Loré, K. Efficient Targeting and Activation of Antigen-Presenting Cells In Vivo after Modified mRNA Vaccine Administration in Rhesus Macaques. Mol. Ther. 2017, 25, 2635– 2647, DOI: 10.1016/j.ymthe.2017.08.006
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Efficient Targeting and Activation of Antigen-Presenting Cells In Vivo after Modified mRNA Vaccine Administration in Rhesus Macaques
Liang, Frank; Lindgren, Gustaf; Lin, Ang; Thompson, Elizabeth A.; Ols, Sebastian; Roehss, Josefine; John, Shinu; Hassett, Kimberly; Yuzhakov, Olga; Bahl, Kapil; Brito, Luis A.; Salter, Hugh; Ciaramella, Giuseppe; Lore, Karin
Molecular Therapy (2017), 25 (12), 2635-2647CODEN: MTOHCK; ISSN:1525-0024. (Cell Press)
MRNA vaccines are rapidly emerging as a powerful platform for infectious diseases because they are well tolerated, immunogenic, and scalable and are built on precise but adaptable antigen design. We show that two immunizations of modified non-replicating mRNA encoding influenza H10 hemagglutinin (HA) and encapsulated in lipid nanoparticles (LNP) induce protective HA inhibition titers and H10-specific CD4+ T cell responses after i.m. or intradermal delivery in rhesus macaques. Administration of LNP/mRNA induced rapid and local infiltration of neutrophils, monocytes, and dendritic cells (DCs) to the site of administration and the draining lymph nodes (LNs). While these cells efficiently internalized LNP, mainly monocytes and DCs translated the mRNA and upregulated key co-stimulatory receptors (CD80 and CD86). This coincided with upregulation of type I IFN-inducible genes, including MX1 and CXCL10. The innate immune activation was transient and resulted in priming of H10-specific CD4+ T cells exclusively in the vaccine-draining LNs. Collectively, this demonstrates that mRNA-based vaccines induce type-I IFN-polarized innate immunity and, when combined with antigen prodn. by antigen-presenting cells, lead to generation of potent vaccine-specific responses.

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Cite this: ACS Nano 2019, 13, 5, 5920–5930

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https://doi.org/10.1021/acsnano.9b01774

Published May 2, 2019

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Abstract
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Scheme 1
Scheme 1. Schematic of Lipid Nanoparticle (LNP) Formulations Used for DoE Analysis: (a) Lipid Nanoparticles Containing a Complexing Lipid, DOPE, and Cholesterol; and (b) Complexing Lipids Used in the DoE Library
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Figure 1
Figure 1. Firefly luciferase expression in human skin explants over the course of 21 days after ID injection of three separate, simultaneous injections of 10 μg of saRNA with a mass ratio of lipid to RNA of 4:1 (w/w): (a) time course of ex vivo imaging of luciferase expressed by replicon RNA delivered with the initial formulation of DOTAP LNPs; and (b) quantification of luciferase expression, expressed as the mean total flux (p/s) ± standard deviation for n = 3.
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Figure 2
Figure 2. Firefly luciferase expression in human skin explants injected intradermally with LNP formulations with varying lipid identity and LNP dose containing 2 μg of saRNA with medium particle concentration (10⁸ particles/mL) at varying ratios of lipid to RNA (w/w): (a) ex vivo imaging of explants after 11 days and (b) quantification of luciferase imagine expressed as the mean total flux (p/s) ± standard deviation for n = 5.
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Figure 3
Figure 3. Firefly luciferase expression in human skin explants injected intradermally with LNP formulations with varying lipid and particle concentrations containing 2 μg of saRNA with a ratio of total lipid to RNA of 90:1 (w/w): (a) ex vivo imaging of explants after 11 days and (b) quantification of luciferase imagine expressed as the mean total flux (p/s) ± standard deviation for n = 5. High and low particle concentrations are defined as 10⁹ and 10⁷ particles/mL, respectively, while high lipid concentration is defined as 7.5 mg/mL. H[L]/H[P] denotes high lipid concentration and high particle concentration; H[L]/L[P] denotes high lipid concentration and low particle concentration.
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Figure 4
Figure 4. Firefly luciferase expression in human skin explants injected intradermally with LNP formulations with varying ratios of cationic and zwitterionic lipids containing 2 μg of saRNA with a total lipid to RNA ratio of 18:1 (w/w) and medium particle concentration (10⁸ particles/mL): (a) ex vivo imaging of explants after 11 days and (b) quantification of luciferase imagine expressed as mean total flux (p/s) ± standard deviation for n = 5.
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Figure 5
Figure 5. Standard least-squares effect screening DoE analysis of lipid nanoparticle formulation in human skin explants.
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Figure 6
Figure 6. Comparison of fold change luciferase expression of tested LNP formulations normalized to original DOTAP formulation (blue bar). Values are expressed fold change total flux (p/s) ± standard deviation.
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Figure 7
Figure 7. GFP expression in human skin cells after intradermal injection with LNP formulations, as determined using flow cytometry: (a) histogram of number of cells expressing GFP for each formulation, and (b) percentage of GFP-positive cells of total live cells for each sample. Bar represents the average ± standard deviation, with a significance of α = 0.05 indicated by an asterisk (*).
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Figure 8
Figure 8. Identity of cells present in human skin explants and GFP+ cells after ID injection of LNP formulations, as determined by flow cytometry: (a) identity of cells in the population of cells extracted from human skin explants, and (b) identity of GFP-expressing skin cells from explants treated with LNP-formulated RNA. The blue sections of each of the small pie charts indicate the total percentage of immune cells in the GFP+ cell population.
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References
This article references 37 other publications.
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  A review. The discovery of Toll-like receptors (TLRs) as components that recognize conserved structures in pathogens has greatly advanced understanding of how the body senses pathogen invasion, triggers innate immune responses and primes antigen-specific adaptive immunity. Although TLRs are crit. for host defense, it has become apparent that loss of neg. regulation of TLR signaling, as well as recognition of self mols. by TLRs, are strongly assocd. with the pathogenesis of inflammatory and autoimmune diseases. Furthermore, it is now clear that the interaction between TLRs and recently identified cytosolic innate immune sensors is crucial for mounting effective immune responses. Here we describe the recent advances that have been made by research into the role of TLR biol. in host defense and disease.
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  An alphavirus replicon particle chimera derived from Venezuelan equine encephalitis and Sindbis viruses is a potent gene-based vaccine delivery vector
  Perri, Silvia; Greer, Catherine E.; Thudium, Kent; Doe, Barbara; Legg, Harold; Liu, Hong; Romero, Raul E.; Tang, Zequn; Bin, Qian; Dubensky, Thomas W., Jr.; Vajdy, Michael; Otten, Gillis R.; Polo, John M.
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  Alphavirus replicon particle-based vaccine vectors derived from Sindbis virus (SIN), Semliki Forest virus, and Venezuelan equine encephalitis virus (VEE) have been shown to induce robust antigen-specific cellular, humoral, and mucosal immune responses in many animal models of infectious disease and cancer. However, since little is known about the relative potencies among these different vectors, we compared the immunogenicity of replicon particle vectors derived from two very different parental alphaviruses, VEE and SIN, expressing a human immunodeficiency virus type 1 p55Gag antigen. Moreover, to explore the potential benefits of combining elements from different alphaviruses, we generated replicon particle chimeras of SIN and VEE. Two distinct strategies were used to produce particles with VEE-p55gag replicon RNA packaged within SIN envelope glycoproteins and SIN-p55gag replicon RNA within VEE envelope glycoproteins. Each replicon particle configuration induced Gag-specific CD8+ T-cell responses in murine models when administered alone or after priming with DNA. However, Gag-specific responses varied dramatically, with the strongest responses to this particular antigen correlating with the VEE replicon RNA, irresp. of the source of envelope glycoproteins. Comparing the replicons with respect to heterologous gene expression levels and sensitivity to alpha/beta interferon in cultured cells indicated that each might contribute to potency differences. This work shows that combining desirable elements from VEE and SIN into a replicon particle chimera may be a valuable approach toward the goal of developing vaccine vectors with optimal in vivo potency, ease of prodn., and safety.
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  Despite more than two decades of research and development on nucleic acid vaccines, there is still no com. product for human use. Taking advantage of the recent innovations in systemic delivery of short interfering RNA (siRNA) using lipid nanoparticles (LNPs), we developed a self-amplifying RNA vaccine. Here we show that nonviral delivery of a 9-kb self-amplifying RNA encapsulated within an LNP substantially increased immunogenicity compared with delivery of unformulated RNA. This unique vaccine technol. was found to elicit broad, potent, and protective immune responses, that were comparable to a viral delivery technol., but without the inherent limitations of viral vectors. Given the many pos. attributes of nucleic acid vaccines, our results suggest that a comprehensive evaluation of nonviral technologies to deliver self-amplifying RNA vaccines is warranted.
10. 10
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  Potent immune responses in rhesus macaques induced by nonviral delivery of a selfamplifying RNA vaccine expressing HIV type 1 envelope with a cationic nanoemulsion
  Bogers, Willy M.; Oostermeijer, Herman; Mooij, Petra; Koopman, Gerrit; Verschoor, Ernst J.; Davis, David; Ulmer, Jeffrey B.; Brito, Luis A.; Cu, Yen; Banerjee, Kaustuv; Otten, Gillis R.; Burke, Brian; Dey, Antu; Heeney, Jonathan L.; Shen, Xiaoying; Tomaras, Georgia D.; Labranche, Celia; Montefiori, David C.; Liao, Hua-Xin; Haynes, Barton; Geall, Andrew J.; Barnett, Susan W.
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  Self-amplifying mRNA (mRNA) of pos.-strand RNA viruses are effective vectors for in situ expression of vaccine antigens and have potential as a new vaccine technol. platform well suited for global health applications. The SAM vaccine platform is based on a synthetic, self-amplifying mRNA delivered by a nonviral delivery system. The safety and immunogenicity of an HIV SAM vaccine encoding a clade C envelope glycoprotein formulated with a cationic nanoemulsion (CNE) delivery system was evaluated in rhesus macaques. The HIV SAM vaccine induced potent cellular immune responses that were greater in magnitude than those induced by self-amplifying mRNA packaged in a viral replicon particle (VRP) or by a recombinant HIV envelope protein formulated with MF59 adjuvant, anti-envelope binding (including anti-V1V2), and neutralizing antibody responses that exceeded those induced by the VRP vaccine. These studies provide the first evidence in nonhuman primates that HIV vaccination with a relatively low dose (50 μg) of formulated self-amplifying mRNA is safe and immunogenic.
11. 11
  Perche, F.; Benvegnu, T.; Berchel, M.; Lebegue, L.; Pichon, C.; Jaffrès, P.-A.; Midoux, P. Enhancement of Dendritic Cells Transfection In Vivo and of Vaccination against B16F10 Melanoma with Mannosylated Histidylated Lipopolyplexes Loaded with Tumor Antigen Messenger RNA. Nanomedicine (N. Y., NY, U. S.) 2011, 7, 445– 453, DOI: 10.1016/j.nano.2010.12.010
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  Systemic delivery of messenger RNA for the treatment of pancreatic cancer using polyplex nanomicelles with a cholesterol moiety
  Uchida, Satoshi; Kinoh, Hiroaki; Ishii, Takehiko; Matsui, Akitsugu; Tockary, Theofilus Agrios; Takeda, Kaori Machitani; Uchida, Hirokuni; Osada, Kensuke; Itaka, Keiji; Kataoka, Kazunori
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  Systemic delivery of mRNA (mRNA) is tech. challenging because mRNA is highly susceptible to enzymic degrdn. in the blood circulation. In this study, we used a nanomicelle-based platform, prepd. from mRNA and poly(ethylene glycol) (PEG)-polycation block copolymers. A cholesterol (Chol) moiety was attached to the ω-terminus of the block copolymer to increase the stability of the nanomicelle by hydrophobic interaction. After in vitro screening, polyaspartamide with four aminoethylene repeats in its side chain (PAsp(TEP)) was selected as the cationic segment of the block copolymer, because it contributes to enhance nuclease resistance and high protein expression from the mRNA. After i.v. injection, PEG-PAsp(TEP)-Chol nanomicelles showed significantly enhanced blood retention of mRNA in comparison to nanomicelles without Chol. We used the nanomicelles for treating intractable pancreatic cancer in a s.c. inoculation mouse model through the delivery of mRNA encoding an anti-angiogenic protein (sFlt-1). PEG-PAsp(TEP)-Chol nanomicelles generated efficient protein expression from the delivered mRNA in tumor tissue, resulting in remarkable inhibition of the tumor growth, whereas nanomicelles without Chol failed to show a detectable therapeutic effect. In conclusion, the stabilized nanomicelle system led to the successful systemic delivery of mRNA in therapeutic application, holding great promise for the treatment of various diseases.
13. 13
  Fotin-Mleczek, M.; Zanzinger, K.; Heidenreich, R.; Lorenz, C.; Kowalczyk, A.; Kallen, K.-J.; Huber, S. M. mRNA-Based Vaccines Synergize with Radiation Therapy to Eradicate Established Tumors. Radiat. Oncol. 2014, 9, 180, DOI: 10.1186/1748-717X-9-180
  13
  mRNA-based vaccines synergize with radiation therapy to eradicate established tumors
  Fotin-Mleczek, Mariola; Zanzinger, Kai; Heidenreich, Regina; Lorenz, Christina; Kowalczyk, Aleksandra; Kallen, Karl-Josef; Huber, Stephan M.
  Radiation Oncology (2014), 9 (), 180/1-180/23CODEN: ROANCM; ISSN:1748-717X. (BioMed Central Ltd.)
  Background: The eradication of large, established tumors by active immunotherapy is a major challenge because of the numerous cancer evasion mechanisms that exist. This study aimed to establish a novel combination therapy consisting of mRNA (mRNA)-based cancer vaccines and radiation, which would facilitate the effective treatment of established tumors with aggressive growth kinetics. Methods: The combination of a tumor-specific mRNA-based vaccination with radiation was tested in two syngeneic tumor models, a highly immunogenic E.G7-OVA and a low immunogenic Lewis lung cancer (LLC). The mol. mechanism induced by the combination therapy was evaluated via gene expression arrays as well as flow cytometry analyses of tumor infiltrating cells. Results: In both tumor models we demonstrated that a combination of mRNA-based immunotherapy with radiation results in a strong synergistic anti-tumor effect. This was manifested as either complete tumor eradication or delay in tumor growth. Gene expression anal. of mouse tumors revealed a variety of substantial changes at the tumor site following radiation. Genes assocd. with antigen presentation, infiltration of immune cells, adhesion, and activation of the innate immune system were upregulated. A combination of radiation and immunotherapy induced significant downregulation of tumor assocd. factors and upregulation of tumor suppressors. Moreover, combination therapy significantly increased CD4+, CD8+ and NKT cell infiltration of mouse tumors. Conclusion: Our data provide a scientific rationale for combining immunotherapy with radiation and provide a basis for the development of more potent anti-cancer therapies.
14. 14
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  14
  Self-replicating Replicon-RNA Delivery to Dendritic Cells by Chitosan-nanoparticles for Translation In Vitro and In Vivo
  McCullough, Kenneth C.; Bassi, Isabelle; Milona, Panagiota; Suter, Rolf; Thomann-Harwood, Lisa; Englezou, Pavlos; Demoulins, Thomas; Ruggli, Nicolas
  Molecular Therapy--Nucleic Acids (2014), 3 (7), e173CODEN: MTAOC5; ISSN:2162-2531. (Nature Publishing Group)
  Self-amplifying replicon RNA (RepRNA) possesses high potential for increasing antigen load within dendritic cells (DCs). The major aim of the present work was to define how RepRNA delivered by biodegradable, chitosan-based nanoparticulate delivery vehicles (nanogel-alginate (NGA)) interacts with DCs, and whether this could lead to translation of the RepRNA in the DCs. Although studies employed virus replicon particles (VRPs), there are no reports on biodegradable, nanoparticulate vehicle delivery of RepRNA. VRP studies employed cytopathogenic agents, contrary to DC requirements-slow processing and antigen retention. We employed noncytopathogenic RepRNA with NGA, demonstrating for the first time the efficiency of RepRNA assocn. with nanoparticles, NGA delivery to DCs, and RepRNA internalization by DCs. RepRNA accumulated in vesicular structures, with patterns typifying cytosolic release. This promoted RepRNA translation, in vitro and in vivo. Delivery and translation were RepRNA concn.-dependent, occurring in a kinetic manner. Including cationic lipids with chitosan during nanoparticle formation enhanced delivery and translation kinetics, but was not required for translation of immunogenic levels in vivo. This work describes for the first time the characteristics assocd. with chitosan-nanoparticle delivery of self-amplifying RepRNA to DCs, leading to translation of encoded foreign genes, namely influenza virus hemagglutinin and nucleoprotein.
15. 15
  Hekele, A.; Bertholet, S.; Archer, J.; Gibson, D. G.; Palladino, G.; Brito, L. A.; Otten, G. R.; Brazzoli, M.; Buccato, S.; Bonci, A.; Casini, D.; Maione, D.; Qi, Z.-Q.; Gill, J. E.; Caiazza, N. C.; Urano, J.; Hubby, B.; Gao, G. F.; Shu, Y.; De Gregorio, E.; Mandl, C. W.; Mason, P. W.; Settembre, E. C.; Ulmer, J. B.; Craig Venter, J.; Dormitzer, P. R.; Rappuoli, R.; Geall, A. J. Rapidly Produced SAM(®) Vaccine against H7N9 Influenza Is Immunogenic in Mice. Emerging Microbes Infect. 2013, 2, 1– 7, DOI: 10.1038/emi.2013.54
  There is no corresponding record for this reference.
16. 16
  Brazzoli, M.; Magini, D.; Bonci, A.; Buccato, S.; Giovani, C.; Kratzer, R.; Zurli, V.; Mangiavacchi, S.; Casini, D.; Brito, L. M.; De Gregorio, E.; Mason, P. W.; Ulmer, J. B.; Geall, A. J.; Bertholet, S. Induction of Broad-Based Immunity and Protective Efficacy by Self-Amplifying mRNA Vaccines Encoding Influenza Virus Hemagglutinin. J. Virol. 2016, 90, 332, DOI: 10.1128/JVI.01786-15
  16
  Induction of broad-based immunity and protective efficacy by selfamplifying mRNA vaccines encoding influenza virus hemagglutinin
  Brazzoli, Michela; Magini, Diletta; Bonci, Alessandra; Buccato, Scilla; Giovani, Cinzia; Kratzer, Roland; Zurli, Vanessa; Mangiavacchi, Simona; Casini, Daniele; Brito, Luis M.; De Gregorio, Ennio; Mason, Peter W.; Ulmer, Jeffrey B.; Geall, Andrew J.; Bertholet, Sylvie
  Journal of Virology (2016), 90 (1), 332-344CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)
  Seasonal influenza is a vaccine-preventable disease that remains a major health problem worldwide, esp. in immunocompromised populations. The impact of influenza disease is even greater when strains drift, and influenza pandemics can result when animal-derived influenza virus strains combine with seasonal strains. In this study, we used the SAM technol. and characterized the immunogenicity and efficacy of a self-amplifying mRNA expressing influenza virus hemagglutinin (HA) antigen [SAM(HA)] formulated with a novel oil-in-water cationic nanoemulsion. We demonstrated that SAM(HA) was immunogenic in ferrets and facilitated containment of viral replication in the upper respiratory tract of influenza virus-infected animals. In mice, SAM(HA) induced potent functional neutralizing antibody and cellular immune responses, characterized by HA-specific CD4 T helper 1 and CD8 cytotoxic T cells. Furthermore, mice immunized with SAM(HA) derived from the influenza A virus A/California/7/2009 (H1N1) strain (Cal) were protected from a lethal challenge with the heterologous mouse-adapted A/PR/8/1934 (H1N1) virus strain (PR8). Sera derived from SAM(H1-Cal)-immunized animals were not cross-reactive with the PR8 virus, whereas cross-reactivity was obsd. for HA-specific CD4 and CD8 T cells. Finally, depletion of T cells demonstrated that T-cell responses were essential in mediating heterologous protection. If the SAM vaccine platform proves safe, well tolerated, and effective in humans, the fully synthetic SAM vaccine technol. could provide a rapid response platform to control pandemic influenza.
17. 17
  Kauffman, K. J.; Dorkin, J. R.; Yang, J. H.; Heartlein, M. W.; DeRosa, F.; Mir, F. F.; Fenton, O. S.; Anderson, D. G. Optimization of Lipid Nanoparticle Formulations for mRNA Delivery In Vivo with Fractional Factorial and Definitive Screening Designs. Nano Lett. 2015, 15, 7300– 7306, DOI: 10.1021/acs.nanolett.5b02497
  17
  Optimization of Lipid Nanoparticle Formulations for mRNA Delivery in Vivo with Fractional Factorial and Definitive Screening Designs
  Kauffman, Kevin J.; Dorkin, J. Robert; Yang, Jung H.; Heartlein, Michael W.; De Rosa, Frank; Mir, Faryal F.; Fenton, Owen S.; Anderson, Daniel G.
  Nano Letters (2015), 15 (11), 7300-7306CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
  Intracellular delivery of mRNA (mRNA) has the potential to induce protein prodn. for many therapeutic applications. Although lipid nanoparticles have shown considerable promise for the delivery of small interfering RNAs (siRNA), their utility as agents for mRNA delivery has only recently been investigated. The most common siRNA formulations contain four components: an amine-contg. lipid or lipid-like material, phospholipid, cholesterol, and lipid-anchored polyethylene glycol, the relative ratios of which can have profound effects on the formulation potency. Here, we develop a generalized strategy to optimize lipid nanoparticle formulations for mRNA delivery to the liver in vivo using Design of Expt. (DOE) methodologies including Definitive Screening and Fractional Factorial Designs. By simultaneously varying lipid ratios and structures, we developed an optimized formulation which increased the potency of erythropoietin-mRNA-loaded C12-200 lipid nanoparticles 7-fold relative to formulations previously used for siRNA delivery. Key features of this optimized formulation were the incorporation of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and increased ionizable lipid:mRNA wt. ratios. Interestingly, the optimized lipid nanoparticle formulation did not improve siRNA delivery, indicating differences in optimized formulation parameter design spaces for siRNA and mRNA. We believe the general method described here can accelerate in vivo screening and optimization of nanoparticle formulations with large multidimensional design spaces.
18. 18
  Bahl, K.; Senn, J. J.; Yuzhakov, O.; Bulychev, A.; Brito, L. A.; Hassett, K. J.; Laska, M. E.; Smith, M.; Almarsson, Ö.; Thompson, J.; Ribeiro, A.; Watson, M.; Zaks, T.; Ciaramella, G. Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9 Influenza Viruses. Mol. Ther. 2017, 25, 1316– 1327, DOI: 10.1016/j.ymthe.2017.03.035
  18
  Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9 Influenza Viruses
  Bahl, Kapil; Senn, Joe J.; Yuzhakov, Olga; Bulychev, Alex; Brito, Luis A.; Hassett, Kimberly J.; Laska, Michael E.; Smith, Mike; Almarsson, Orn; Thompson, James; Ribeiro, Amilcar; Watson, Mike; Zaks, Tal; Ciaramella, Giuseppe
  Molecular Therapy (2017), 25 (6), 1316-1327CODEN: MTOHCK; ISSN:1525-0024. (Cell Press)
  Recently, the World Health Organization confirmed 120 new human cases of avian H7N9 influenza in China resulting in 37 deaths, highlighting the concern for a potential pandemic and the need for an effective, safe, and high-speed vaccine prodn. platform. Prodn. speed and scale of mRNA-based vaccines make them ideally suited to impede potential pandemic threats. Here we show that lipid nanoparticle (LNP)-formulated, modified mRNA vaccines, encoding hemagglutinin (HA) proteins of H10N8 (A/Jiangxi-Donghu/346/2013) or H7N9 (A/Anhui/1/2013), generated rapid and robust immune responses in mice, ferrets, and nonhuman primates, as measured by hemagglutination inhibition (HAI) and microneutralization (MN) assays. A single dose of H7N9 mRNA protected mice from a lethal challenge and reduced lung viral titers in ferrets. Interim results from a first-in-human, escalating-dose, phase 1 H10N8 study show very high seroconversion rates, demonstrating robust prophylactic immunity in humans. Adverse events (AEs) were mild or moderate with only a few severe and no serious events. These data show that LNP-formulated, modified mRNA vaccines can induce protective immunogenicity with acceptable tolerability profiles.
19. 19
  van den Berg, J. H.; Nuijen, B.; Beijnen, J. H.; Vincent, A.; van Tinteren, H.; Kluge, J.; Woerdeman, L. A. E.; Hennink, W. E.; Storm, G.; Schumacher, T. N.; Haanen, J. B. A. G. Optimization of Intradermal Vaccination by DNA Tattooing in Human Skin. Hum. Gene Ther. 2009, 20, 181– 189, DOI: 10.1089/hum.2008.073
  19
  Optimization of Intradermal Vaccination by DNA Tattooing in Human Skin
  van den Berg, Joost H.; Nuijen, Bastiaan; Beijnen, Jos H.; Vincent, Andrew; van Tinteren, Harm; Kluge, Joern; Woerdeman, Leonie A. E.; Hennink, Wim E.; Storm, Gert; Schumacher, Ton N.; Haanen, John B. A. G.
  Human Gene Therapy (2009), 20 (3), 181-189CODEN: HGTHE3; ISSN:1043-0342. (Mary Ann Liebert, Inc.)
  The intradermal administration of DNA vaccines by tattooing is a promising delivery technique for genetic immunization, with proven high immunogenicity in mice and in nonhuman primates. However, the parameters that result in optimal expression of DNA vaccines that are applied by this strategy to human skin are currently unknown. To address this issue we set up an ex vivo human skin model in which DNA vaccine-induced expression of reporter proteins could be monitored longitudinally. Using this model we demonstrate the following: First, the vast majority of cells that express DNA vaccine-encoded antigen in human skin are formed by epidermal keratinocytes, with only a small fraction (about 1%) of antigen-pos. epidermal Langerhans cells. Second, using full randomization of DNA tattoo variables we show that an increase in DNA concn., needle depth, and tattoo time all significantly increase antigen expression (p < 0.001), with DNA concn. forming the most crit. variable influencing the level of antigen expression. Finally, in spite of the marked immunogenicity of this vaccination method in animal models, transfection efficiency of the technique is shown to be extremely low, estd. at approx. 2 to 2000 out of 1 × 1010 copies of plasmid applied. This finding, coupled with the obsd. dependency of antigen expression on DNA concn., suggests that the development of strategies that can enhance in vivo transfection efficacy would be highly valuable. Collectively, this study shows that an ex vivo human skin model can be used to det. the factors that control vaccine-induced antigen expression and define the optimal parameters for the evaluation of DNA tattoo or other dermal delivery techniques in phase 1 clin. trials.
20. 20
  Gelfant, S. Of Mice and Men” the Cell Cycle in Human Epidermis In Vivo. J. Invest. Dermatol. 1982, 78, 296– 299, DOI: 10.1111/1523-1747.ep12507367
  20
  "Of mice and men" the cell cycle in human epidermis in vivo
  Gelfant S
  The Journal of investigative dermatology (1982), 78 (4), 296-9 ISSN:0022-202X.
  This article deals with and compares cell cycle information obtained in mouse and in human epidermis in vivo. In order to compare data in mouse and in man, DNA labeling and mitotic index experiments were performed to obtain cell cycle information in normal human epidermis in vivo. Experiments were also performed on genetically inbred and outbred strains of mice--to provide a clue to the differences observed between mouse and man. The article makes the following points: 1. In contrast to mouse epidermis, there are no consistent diurnal fluctuations in and there is no cell kinetic relationship between mitotic and DNA-labeling indices in normal human epidermis in vivo. The variability from individual to individual in human subjects and the lack of cell cycle-related circadian fluctuations, preclude the use of statistical analysis and the use of conventional cell kinetic principles in understanding epidermal cell proliferation in man and may preclude the use of circadian rhythmicity for therapy scheduling. 2. The consistent and intelligible cell cycle information obtained in laboratory mice (as compared to man) is not due to the genetically inbred condition of mice. 3. This report introduces the use of ambient temperature as a potential nontoxic cell cycle tool for manipulating epidermal cell proliferation in the therapy of human proliferative skin diseases.
21. 21
  Love, K. T.; Mahon, K. P.; Levins, C. G.; Whitehead, K. A.; Querbes, W.; Dorkin, J. R.; Qin, J.; Cantley, W.; Qin, L. L.; Racie, T.; Frank-Kamenetsky, M.; Yip, K. N.; Alvarez, R.; Sah, D. W. Y.; de Fougerolles, A.; Fitzgerald, K.; Koteliansky, V.; Akinc, A.; Langer, R.; Anderson, D. G. Lipid-Like Materials for Low-Dose, In Vivo Gene Silencing. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 1864– 1869, DOI: 10.1073/pnas.0910603106
  21
  Lipid-like materials for low-dose, in vivo gene silencing
  Love, Kevin T.; Mahon, Kerry P.; Levins, Christopher G.; Whitehead, Kathryn A.; Querbes, William; Dorkin, J. Robert; Qin, June; Cantley, William; Qin, Liu Liang; Racie, Timothy; Frank-Kamenetsky, Maria; Yip, Ka Ning; Alvarez, Rene; Sah, Dinah W. Y.; de Fougerolles, Antonin; Fitzgerald, Kevin; Koteliansky, Victor; Akinc, Akin; Langer, Robert; Anderson, Daniel G.
  Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (5), 1864-1869, S1864/1-S1864/6CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  Significant effort has been applied to discover and develop vehicles which can guide small interfering RNAs (siRNA) through the many barriers guarding the interior of target cells. While studies have demonstrated the potential of gene silencing in vivo, improvements in delivery efficacy are required to fulfill the broadest potential of RNA interference therapeutics. Through the combinatorial synthesis and screening of a different class of materials, a formulation has been identified that enables siRNA-directed liver gene silencing in mice at doses below 0.01 mg/kg. This formulation was also shown to specifically inhibit expression of five hepatic genes simultaneously, after a single injection. The potential of this formulation was further validated in nonhuman primates, where high levels of knockdown of the clin. relevant gene transthyretin was obsd. at doses as low as 0.03 mg/kg. To our knowledge, this formulation facilitates gene silencing at orders-of-magnitude lower doses than required by any previously described siRNA liver delivery system.
22. 22
  Ball, R. L.; Hajj, K. A.; Vizelman, J.; Bajaj, P.; Whitehead, K. A. Lipid Nanoparticle Formulations for Enhanced Co-Delivery of siRNA and mRNA. Nano Lett. 2018, 18, 3814– 3822, DOI: 10.1021/acs.nanolett.8b01101
  22
  Lipid nanoparticle formulations for enhanced co-delivery of siRNA and mRNA
  Ball, Rebecca L.; Hajj, Khalid A.; Vizelman, Jamie; Bajaj, Palak; Whitehead, Kathryn A.
  Nano Letters (2018), 18 (6), 3814-3822CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
  Although mRNA and siRNA have significant therapeutic potential, their simultaneous delivery has not been previously explored. To facilitate the treatment of diseases assocd. with aberrant gene upregulation and downregulation, we sought to co-formulate siRNA and mRNA in a single lipidoid nanoparticle (LNP) formulation. We accommodated the distinct mol. characteristics of mRNA and siRNA in a formulation consisting of an ionizable and biodegradable amine-contg. lipidoid, cholesterol, DSPC, DOPE, and PEG-lipid. Surprisingly, the co-formulation of siRNA and mRNA in the same LNP enhanced the efficacy of both drugs in vitro and in vivo. Compared to LNPs encapsulating siRNA only, co-formulated LNPs improved Factor VII gene silencing in mice from 44 to 87% at an siRNA dose of 0.03 mg/kg. Co-formulation also improved mRNA delivery, as a 0.5 mg/kg dose of mRNA co-formulated with siRNA induced three times the luciferase protein expression compared to when siRNA was not included. As not all gene therapy applications require both RNA drugs, we sought to extend the benefit of co-formulated LNPs to formulations encapsulating only a single type of RNA. We accomplished this by substituting the "helper" RNA with a neg. charged polymer, polystyrenesulfonate (PSS). LNPs contg. PSS mediated the same level of protein silencing or expression as std. LNPs using 2-3-fold less RNA. For example, LNPs formulated with and without PSS induced 50% Factor VII silencing at siRNA doses of 0.01 and 0.03 mg/kg, resp. Together, these studies demonstrate potent co-delivery of siRNA and mRNA and show that inclusion of a neg. charged "helper polymer" enhances the efficacy of LNP delivery systems.
23. 23
  Henriksen-Lacey, M.; Christensen, D.; Bramwell, V. W.; Lindenstrøm, T.; Agger, E. M.; Andersen, P.; Perrie, Y. Comparison of the Depot Effect and Immunogenicity of Liposomes Based on Dimethyldioctadecylammonium (DDA), 3β-[N-(N′,N′-Dimethylaminoethane)Carbomyl] Cholesterol (DC-Chol), and 1,2-Dioleoyl-3-Trimethylammonium Propane (DOTAP): Prolonged Liposome Retention Mediates Stronger Th1 Responses. Mol. Pharmaceutics 2011, 8, 153– 161, DOI: 10.1021/mp100208f
  23
  Comparison of the Depot Effect and Immunogenicity of Liposomes Based on Dimethyldioctadecylammonium (DDA), 3β-[N-(N',N'-Dimethylaminoethane)carbomyl] Cholesterol (DC-Chol), and 1,2-Dioleoyl-3-trimethylammonium Propane (DOTAP): Prolonged Liposome Retention Mediates Stronger Th1 Responses
  Henriksen-Lacey, Malou; Christensen, Dennis; Bramwell, Vincent W.; Lindenstrom, Thomas; Agger, Else Marie; Andersen, Peter; Perrie, Yvonne
  Molecular Pharmaceutics (2011), 8 (1), 153-161CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)
  The immunostimulatory capacities of cationic liposomes are well-documented and are attributed both to inherent immunogenicity of the cationic lipid and more phys. capacities such as the formation of antigen depots and antigen delivery. Very few studies have however been conducted comparing the immunostimulatory capacities of different cationic lipids. In the present study we therefore chose to investigate three of the most well-known cationic liposome-forming lipids as potential adjuvants for protein subunit vaccines. The ability of 3β-[N-(N',N'-dimethylaminoethane)carbomyl] cholesterol (DC-Chol), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), and dimethyldioctadecylammonium (DDA) liposomes incorporating immunomodulating trehalose dibehenate (TDB) to form an antigen depot at the site of injection (SOI) and to induce immunol. recall responses against coadministered tuberculosis vaccine antigen Ag85B-ESAT-6 are reported. Furthermore, phys. characterization of the liposomes is presented. Our results suggest that liposome compn. plays an important role in vaccine retention at the SOI and the ability to enable the immune system to induce a vaccine specific recall response. While all three cationic liposomes facilitated increased antigen presentation by antigen presenting cells, the monocyte infiltration to the SOI and the prodn. of IFN-γ upon antigen recall was markedly higher for DDA and DC-Chol based liposomes which exhibited a longer retention profile at the SOI. A long-term retention and slow release of liposome and vaccine antigen from the injection site hence appears to favor a stronger Th1 immune response.
24. 24
  De Serrano, L. O.; Burkhart, D. J. Liposomal Vaccine Formulations as Prophylactic Agents: Design Considerations for Modern Vaccines. J. Nanobiotechnol. 2017, 15, 83, DOI: 10.1186/s12951-017-0319-9
  24
  Liposomal vaccine formulations as prophylactic agents: design considerations for modern vaccines
  De Serrano, Luis O.; Burkhart, David J.
  Journal of Nanobiotechnology (2017), 15 (), 83/1-83/23CODEN: JNOAAO; ISSN:1477-3155. (BioMed Central Ltd.)
  A review. Vaccinol. is one of the most important cornerstones in modern medicine, providing better quality of life. The human immune system is composed of innate and adaptive immune processes that interplay when infection occurs. Innate immunity relies on pathogen-assocd. mol. patterns which are recognized by pathogen recognition receptors localized in antigen presenting cells. After antigen processing and presentation, CD4+ T cell polarization occurs, further leading to B cell and CD8+ activation and humoral and cell-mediated adaptive immune responses. Liposomes are being employed as vaccine technologies and their design is of importance to ensure proper immune responses. Physicochem. parameters like liposome size, charge, lamellarity and bilayer fluidity must be completely understood to ensure optimal vaccine stability and efficacy. Liposomal vaccines can be developed to target specific immune cell types for the induction of certain immune responses. In this review, we will present promising liposomal vaccine approaches for the treatment of important viral, bacterial, fungal and parasitic infections (including tuberculosis, TB). Cationic liposomes are the most studied liposome types due to their enhanced interaction with the neg. charged immune cells. Thus, a special section on the cationic lipid dimethyldioctadecylammonium and TB is also presented.
25. 25
  Martino, S.; di Girolamo, I.; Tiribuzi, R.; D’Angelo, F.; Datti, A.; Orlacchio, A. Efficient siRNA Delivery by the Cationic Liposome DOTAP in Human Hematopoietic Stem Cells Differentiating into Dendritic Cells. J. Biomed. Biotechnol. 2009, 2009, 410260, DOI: 10.1155/2009/410260
  25
  Efficient siRNA delivery by the cationic liposome DOTAP in human hematopoietic stem cells differentiating into dendritic cells
  Martino Sabata; di Girolamo Ilaria; Tiribuzi Roberto; D'Angelo Francesco; Datti Alessandro; Orlacchio Aldo
  Journal of biomedicine & biotechnology (2009), 2009 (), 410260 ISSN:.
  RNA interference technology is an ideal strategy to elucidate the mechanisms associated with human CD34(+) hematopoietic stem cell differentiation into dendritic cells. Simple manipulations in vitro can unequivocally yield alloreactive or tolerogenic populations, suggesting key implications of biochemical players that might emerge as therapeutic targets for cancer or graft-versus-host disease. To knockdown proteins typically involved in the biology of dendritic cells, we employed an siRNA delivery system based on the cationic liposome DOTAP as the carrier. Freshly-isolated CD34(+) cells were transfected with siRNA for cathepsin S with negligible cytotoxicity and transfection rates (>60%) comparable to the efficiency shown by lentiviral vectors. Further, cathepsin S knockdown was performed during both cell commitment and through the entire 14-day differentiation process with repeated transfection rounds that had no effect per se on cell development. Tested in parallel, other commercially-available chemical reagents failed to meet acceptable standards. In addition to safe and practical handling, a direct advantage of DOTAP over viral-mediated techniques is that transient silencing effects can be dynamically appraised through the recovery of targeted proteins. Thus, our findings identify DOTAP as an excellent reagent for gene silencing in resting and differentiating CD34(+) cells, suggesting a potential for applications in related preclinical models.
26. 26
  Kaneda, M. M.; Sasaki, Y.; Lanza, G. M.; Milbrandt, J.; Wickline, S. A. Mechanisms of Nucleotide Trafficking During siRNA Delivery to Endothelial Cells Using Perfluorocarbon Nanoemulsions. Biomaterials 2010, 31, 3079– 3086, DOI: 10.1016/j.biomaterials.2010.01.006
  26
  Mechanisms of nucleotide trafficking during siRNA delivery to endothelial cells using perfluorocarbon nanoemulsions
  Kaneda, Megan M.; Sasaki, Yo; Lanza, Gregory M.; Milbrandt, Jeffrey; Wickline, Samuel A.
  Biomaterials (2010), 31 (11), 3079-3086CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
  RNA interference (RNAi) is a useful in vitro research tool, but its application as a safe and effective therapeutic agent may benefit from improved understanding of mechanisms of exogenous siRNA delivery, including cell trafficking and sorting patterns. We report the development of a transfection reagent for siRNA delivery which employs a distinctive non-digestive mode of particle-cell membrane interaction through the formation of a hemifusion complex resulting in lipid raft transport of cargo to the cytosol, bypassing the usual endosomal nanoparticle uptake pathway. We further demonstrate markedly enhanced efficacy over conventional transfection agents for suppressing endothelial cell expression of upregulated vascular adhesion mols.
27. 27
  Zhang, X.; Li, B.; Luo, X.; Zhao, W.; Jiang, J.; Zhang, C.; Gao, M.; Chen, X.; Dong, Y. Biodegradable Amino-Ester Nanomaterials for Cas9 mRNA Delivery In Vitro and In Vivo. ACS Appl. Mater. Interfaces 2017, 9, 25481– 25487, DOI: 10.1021/acsami.7b08163
  27
  Biodegradable Amino-Ester Nanomaterials for Cas9 mRNA Delivery in Vitro and in Vivo
  Zhang, Xinfu; Li, Bin; Luo, Xiao; Zhao, Weiyu; Jiang, Justin; Zhang, Chengxiang; Gao, Min; Chen, Xiaofang; Dong, Yizhou
  ACS Applied Materials & Interfaces (2017), 9 (30), 25481-25487CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
  Efficient and safe delivery of the CRISPR/Cas system is one of the key challenges for genome-editing applications in humans. Herein, we designed and synthesized a series of biodegradable lipidlike compds. contg. ester groups for the delivery of mRNA-encoding Cas9. Two lead materials, termed N-methyl-1,3-propanediamine (MPA)-A and MPA-Ab, showed a tunable rate of biodegrdn. MPA-A with linear ester chains was degraded dramatically faster than MPA-Ab with branched ester chains in the presence of esterase or in wild-type mice. Most importantly, MPA-A and MPA-Ab demonstrated efficient delivery of Cas9 mRNA both in vitro and in vivo. Consequently, these biodegradable lipidlike nanomaterials merit further development as genome-editing delivery tools for biol. and therapeutic applications.
28. 28
  Kim, B.-K.; Hwang, G.-B.; Seu, Y.-B.; Choi, J.-S.; Jin, K. S.; Doh, K.-O. DOTAP/DOPE Ratio and Cell Type Determine Transfection Efficiency with DOTAP-Liposomes. Biochim. Biophys. Acta, Biomembr. 2015, 1848, 1996– 2001, DOI: 10.1016/j.bbamem.2015.06.020
  28
  DOTAP/DOPE ratio and cell type determine transfection efficiency with DOTAP-liposomes
  Kim, Bieong-Kil; Hwang, Guen-Bae; Seu, Young-Bae; Choi, Jong-Soo; Jin, Kyeong Sik; Doh, Kyung-Oh
  Biochimica et Biophysica Acta, Biomembranes (2015), 1848 (10_Part_A), 1996-2001CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
  The effects of lipid compns. on their physicochem. properties and transfection efficiencies were investigated. Four liposome formulations with different 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) to dioleoylphosphatidylethanolamine (DOPE) wt. ratios were investigated, i.e., wt. ratios 1:0 (T1P0), 3:1 (T3P1), 1:1 (T1P1), and 1:3 (T1P3). Mean sizes of liposomes were influenced by their lipid compn. and the prepn. concn. at the time of sonication. Zeta potentials of liposomes were inversely correlated with their liposome sizes. However, neither liposome sizes nor zeta potentials were correlated with transfection efficiency. The optimum compn. of liposomes was cell-line dependent (T1P0 and T3P1 for Huh7 and AGS, T3P1 and T1P1 for COS7, and T1P1 and T1P3 for A549). The shape of lipoplexes was changed from lamellar to inverted hexagonal structure according to the increased ratio of DOPE, but there was no definite advantage of specific structure in transfection efficiency throughout all used cell lines. However, cellular internalization was consistently faster in T1P0, T3P1, T1P1 compared to T1P3 in all cell lines, suggesting the importance of endosomal escape. Our findings show that the transfection efficiency of DOTAP liposomes is mainly influenced by lipid compn. and cell type, and not by size or zeta potential.
29. 29
  Mével, M.; Haudebourg, T.; Colombani, T.; Peuziat, P.; Dallet, L.; Chatin, B.; Lambert, O.; Berchel, M.; Montier, T.; Jaffrès, P.-A.; Lehn, P.; Pitard, B. Important Role of Phosphoramido Linkage in Imidazole-Based Dioleyl Helper Lipids for Liposome Stability and Primary Cell Transfection. J. Gene Med. 2016, 18, 3– 15, DOI: 10.1002/jgm.2869
  29
  Important role of phosphoramido linkage in imidazole-based dioleyl helper lipids for liposome stability and primary cell transfection
  Mevel, Mathieu; Haudebourg, Thomas; Colombani, Thibault; Peuziat, Pauline; Dallet, Laurence; Chatin, Benoit; Lambert, Olivier; Berchel, Mathieu; Montier, Tristan; Jaffres, Paul-Alain; Lehn, Pierre; Pitard, Bruno
  Journal of Gene Medicine (2016), 18 (1-3), 3-15CODEN: JGMEFG; ISSN:1521-2254. (Wiley-Blackwell)
  Background : To optimize synthetic gene delivery systems, there is a need to develop more efficient lipid formulations. Most cationic lipid formulations contain 'helper' neutral lipids because of their ability to increase DNA delivery, in particular by improving endosomal escape of DNA mols. via the pH-buffering effect of protonatable groups and/or fusion with the lipid bilayer of endosomes. Methods : We evaluated the influence of the linker structure between the two oleyl chains in the helper lipid on transfection efficiency in cell lines, as well as in primary cells (hepatocytes/cardiomyocytes). We reported the synthesis of two new pH-buffering imidazole helper lipids characterized by a polar headgroup contg. one (compd. 6) or two (compd. 5) imidazole groups and two oleyl chains linked by an amide group. We studied their assocn. with the aminoglycoside lipidic deriv. dioleylsuccinylparomomycin (DOSP), which contains two oleyl chains linked to the aminoglycoside polar headgroup via an amide function. We compared the morphol. and transfection properties of such binary liposomes of DOSP/5 and DOSP/6 with those of liposomes combining DOSP with another imidazole-based dioleyl helper lipid (MM27) in which a phosphoramido group acts as a linker between the two oleyl chains and imidazole function. Results : The phosphoramido linker in the helper lipid induces a major difference in terms of morphol. and resistance to decomplexation at phys. pH for DOSP/helper lipid complexes. Conclusions : This hybrid dioleyl linker compn. of DOSP/MM27 led to higher transfection efficiency in cell lines and in primary cells compared to complexes with homogeneous dioleyl linker. Copyright © 2015 John Wiley & Sons, Ltd.
30. 30
  Cavalcanti, R. R. M.; Lira, R. B.; Riske, K. A. Study of the Fusion Mechanism of Fusogenic Cationic Liposomes with Anionic Model Membranes. Biophys. J. 2018, 114, 606a, DOI: 10.1016/j.bpj.2017.11.3314
  There is no corresponding record for this reference.
31. 31
  Démoulins, T.; Milona, P.; Englezou, P. C.; Ebensen, T.; Schulze, K.; Suter, R.; Pichon, C.; Midoux, P.; Guzmán, C. A.; Ruggli, N.; McCullough, K. C. Polyethylenimine-Based Polyplex Delivery of Self-Replicating RNA Vaccines. Nanomedicine (N. Y., NY, U. S.) 2016, 12, 711– 722, DOI: 10.1016/j.nano.2015.11.001
  31
  Polyethylenimine-based polyplex delivery of self-replicating RNA vaccines
  Demoulins, Thomas; Milona, Panagiota; Englezou, Pavlos C.; Ebensen, Thomas; Schulze, Kai; Suter, Rolf; Pichon, Chantal; Midoux, Patrick; Guzman, Carlos A.; Ruggli, Nicolas; McCullough, Kenneth C.
  Nanomedicine (New York, NY, United States) (2016), 12 (3), 711-722CODEN: NANOBF; ISSN:1549-9634. (Elsevier)
  Self-amplifying replicon RNA (RepRNA) are large mols. (12-14 kb); their self-replication amplifies mRNA template nos., affording several rounds of antigen prodn., effectively increasing vaccine antigen payloads. Their sensitivity to RNase-sensitivity and inefficient uptake by dendritic cells (DCs) - abs. requirements for vaccine design - were tackled by condensing RepRNA into synthetic, nanoparticulate, polyethylenimine (PEI)-polyplex delivery vehicles. Polyplex-delivery formulations for small RNA mols. cannot be transferred to RepRNA due to its greater size and complexity; the N:P charge ratio and impact of RepRNA folding would influence polyplex condensation, post-delivery decompaction and the cytosolic release essential for RepRNA translation. Polyplex-formulations proved successful for delivery of RepRNA encoding influenza virus hemagglutinin and nucleocapsid to DCs. Cytosolic translocation was facilitated, leading to RepRNA translation. This efficacy was confirmed in vivo, inducing both humoral and cellular immune responses. Accordingly, this paper describes the first PEI-polyplexes providing efficient delivery of the complex and large, self-amplifying RepRNA vaccines.
32. 32
  Pardi, N.; Parkhouse, K.; Kirkpatrick, E.; McMahon, M.; Zost, S. J.; Mui, B. L.; Tam, Y. K.; Karikó, K.; Barbosa, C. J.; Madden, T. D.; Hope, M. J.; Krammer, F.; Hensley, S. E.; Weissman, D. Nucleoside-Modified mRNA Immunization Elicits Influenza Virus Hemagglutinin Stalk-Specific Antibodies. Nat. Commun. 2018, 9, 3361, DOI: 10.1038/s41467-018-05482-0
  32
  Nucleoside-modified mRNA immunization elicits influenza virus hemagglutinin stalk-specific antibodies
  Pardi Norbert; Weissman Drew; Parkhouse Kaela; Zost Seth J; Hensley Scott E; Kirkpatrick Ericka; McMahon Meagan; Krammer Florian; Kirkpatrick Ericka; Mui Barbara L; Tam Ying K; Barbosa Christopher J; Madden Thomas D; Hope Michael J; Kariko Katalin
  Nature communications (2018), 9 (1), 3361 ISSN:.
  Currently available influenza virus vaccines have inadequate effectiveness and are reformulated annually due to viral antigenic drift. Thus, development of a vaccine that confers long-term protective immunity against antigenically distant influenza virus strains is urgently needed. The highly conserved influenza virus hemagglutinin (HA) stalk represents one of the potential targets of broadly protective/universal influenza virus vaccines. Here, we evaluate a potent broadly protective influenza virus vaccine candidate that uses nucleoside-modified and purified mRNA encoding full-length influenza virus HA formulated in lipid nanoparticles (LNPs). We demonstrate that immunization with HA mRNA-LNPs induces antibody responses against the HA stalk domain of influenza virus in mice, rabbits, and ferrets. The HA stalk-specific antibody response is associated with protection from homologous, heterologous, and heterosubtypic influenza virus infection in mice.
33. 33
  Reichmuth, A. M.; Oberli, M. A.; Jaklenec, A.; Langer, R.; Blankschtein, D. mRNA Vaccine Delivery Using Lipid Nanoparticles. Ther. Delivery 2016, 7, 319– 334, DOI: 10.4155/tde-2016-0006
  33
  mRNA vaccine delivery using lipid nanoparticles
  Reichmuth, Andreas M.; Oberli, Matthias A.; Jeklenec, Ana; Langer, Robert; Blankschtein, Daniel
  Therapeutic Delivery (2016), 7 (5), 319-334CODEN: TDHEA7; ISSN:2041-5990. (Future Science Ltd.)
  MRNA vaccines elicit a potent immune response including antibodies and cytotoxic T cells. MRNA vaccines are currently evaluated in clin. trials for cancer immunotherapy applications, but also have great potential as prophylactic vaccines. Efficient delivery of mRNA vaccines will be key for their success and translation to the clinic. Among potential nonviral vectors, lipid nanoparticles are particularly promising. Indeed, lipid nanoparticles can be synthesized with relative ease in a scalable manner, protect the mRNA against degrdn., facilitate endosomal escape, can be targeted to the desired cell type by surface decoration with ligands, and as needed, can be codelivered with adjuvants.
34. 34
  Albanese, A.; Tang, P. S.; Chan, W. C. W. The Effect of Nanoparticle Size, Shape, and Surface Chemistry on Biological Systems. Annu. Rev. Biomed. Eng. 2012, 14, 1– 16, DOI: 10.1146/annurev-bioeng-071811-150124
  34
  The effect of nanoparticle size, shape, and surface chemistry on biological systems
  Albanese, Alexandre; Tang, Peter S.; Chan, Warren C. W.
  Annual Review of Biomedical Engineering (2012), 14 (), 1-16CODEN: ARBEF7; ISSN:1523-9829. (Annual Reviews Inc.)
  A review. An understanding of the interactions between nanoparticles and biol. systems is of significant interest. Studies aimed at correlating the properties of nanomaterials such as size, shape, chem. functionality, surface charge, and compn. with biomol. signaling, biol. kinetics, transportation, and toxicity in both cell culture and animal expts. are under way. These fundamental studies will provide a foundation for engineering the next generation of nanoscale devices. Here, the authors provide rationales for these studies, review the current progress in studies of the interactions of nanomaterials with biol. systems, and provide a perspective on the long-term implications of these findings.
35. 35
  Golestani, R.; Pourfathollah, A. A.; Moazzeni, S. M. An Extreme Strategy for the Production of Hybridoma. Hybridoma 2009, 28, 139– 144, DOI: 10.1089/hyb.2008.0076
  35
  An Extreme Strategy for the Production of Hybridoma
  Golestani, Reza; Pourfathollah, Ali Akbar; Moazzeni, Seyed Mohammad
  Hybridoma (2009), 28 (2), 139-144CODEN: HYBRAV; ISSN:1554-0014. (Mary Ann Liebert, Inc.)
  The ethical issues surrounding human immunization hamper the prodn. of human monoclonal antibody through scarcity of immunized B cells in peripheral blood. This defect can be compensated in part by improvement of hybridoma prodn. techniques. We have developed a new strategy to bypass the toxic effects of polyethylene glycol (PEG) as fusogenic reagent and hypoxanthine aminoptrin thymidine (HAT) as selective medium on newly fused cells. The Epstein-Barr virus (EBV) transformed peripheral blood mononuclear cells (PBMC) of accidentally Rh antigen sensitized persons were fused using cephalin as fugenic reagent, with emetine and actinomycin D pretreated heteromyeloma cells. Our results showed that 19-34% of EBV-transformed B cells were grown as hybridoma clones following selection. This extreme improvement in hybridoma prodn. rate may end the fusion efficiency problem and make hybridoma prodn. a plug-and-play technol.
36. 36
  Phua, K. K. L.; Leong, K. W.; Nair, S. K. Transfection Efficiency and Transgene Expression Kinetics of mRNA Delivered in Naked and Nanoparticle Format. J. Controlled Release 2013, 166, 227– 233, DOI: 10.1016/j.jconrel.2012.12.029
  36
  Transfection efficiency and transgene expression kinetics of mRNA delivered in naked and nanoparticle format
  Phua, Kyle K. L.; Leong, Kam W.; Nair, Smita K.
  Journal of Controlled Release (2013), 166 (3), 227-233CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
  Transfection efficiencies and transgene expression kinetics of mRNA, an emerging class of nucleic acid-based therapeutics, were poorly characterized. In this study, the authors evaluated transfection efficiencies of mRNA delivered in naked and nanoparticle format in vitro and in vivo using GFP and luciferase as reporters. While mRNA nanoparticles transfect primary human and mouse dendritic cells (DCs) efficiently in vitro, naked mRNA could not produce any detectable gene product. The protein expression of nanoparticle-mediated transfection in vitro peaks rapidly within 5-7 h and decays in a biphasic manner. In vivo, naked mRNA is more efficient than mRNA nanoparticles when administered s.c. In contrast, mRNA nanoparticle performs better when administered intranasally and i.v. Gene expression is most transient when delivered i.v. in nanoparticle format with an apparent half-life of 1.4 h and lasts less than 24 h, and most sustained when delivered in the naked format s.c. at the base of tail with an apparent half-life of 18 h and persists for at least 6 days. Notably, exponential decreases in protein expression are consistently obsd. post-delivery of mRNA in vivo regardless of the mode of delivery (naked or nanoparticle) or the site of administration. This study elucidates the performance of mRNA transfection and suggests a niche for mRNA therapeutics when predictable in vivo transgene expression kinetics is imperative.
37. 37
  Liang, F.; Lindgren, G.; Lin, A.; Thompson, E. A.; Ols, S.; Röhss, J.; John, S.; Hassett, K.; Yuzhakov, O.; Bahl, K.; Brito, L. A.; Salter, H.; Ciaramella, G.; Loré, K. Efficient Targeting and Activation of Antigen-Presenting Cells In Vivo after Modified mRNA Vaccine Administration in Rhesus Macaques. Mol. Ther. 2017, 25, 2635– 2647, DOI: 10.1016/j.ymthe.2017.08.006
  37
  Efficient Targeting and Activation of Antigen-Presenting Cells In Vivo after Modified mRNA Vaccine Administration in Rhesus Macaques
  Liang, Frank; Lindgren, Gustaf; Lin, Ang; Thompson, Elizabeth A.; Ols, Sebastian; Roehss, Josefine; John, Shinu; Hassett, Kimberly; Yuzhakov, Olga; Bahl, Kapil; Brito, Luis A.; Salter, Hugh; Ciaramella, Giuseppe; Lore, Karin
  Molecular Therapy (2017), 25 (12), 2635-2647CODEN: MTOHCK; ISSN:1525-0024. (Cell Press)
  MRNA vaccines are rapidly emerging as a powerful platform for infectious diseases because they are well tolerated, immunogenic, and scalable and are built on precise but adaptable antigen design. We show that two immunizations of modified non-replicating mRNA encoding influenza H10 hemagglutinin (HA) and encapsulated in lipid nanoparticles (LNP) induce protective HA inhibition titers and H10-specific CD4+ T cell responses after i.m. or intradermal delivery in rhesus macaques. Administration of LNP/mRNA induced rapid and local infiltration of neutrophils, monocytes, and dendritic cells (DCs) to the site of administration and the draining lymph nodes (LNs). While these cells efficiently internalized LNP, mainly monocytes and DCs translated the mRNA and upregulated key co-stimulatory receptors (CD80 and CD86). This coincided with upregulation of type I IFN-inducible genes, including MX1 and CXCL10. The innate immune activation was transient and resulted in priming of H10-specific CD4+ T cells exclusively in the vaccine-draining LNs. Collectively, this demonstrates that mRNA-based vaccines induce type-I IFN-polarized innate immunity and, when combined with antigen prodn. by antigen-presenting cells, lead to generation of potent vaccine-specific responses.
Supporting Information
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.9b01774.
- LNP characterization of particle size and surface charge (Figure S1); eGFP expression gating strategy (Figure S2); skin explant cell viability (Figure S3); identity of cells present in total population and GFP+ cells (Figure S4); cell viability of human skin explants after LNP-formulation saRNA injection (Figure S5); antibodies used for flow cytometry (Table S1) (PDF)
- nn9b01774_si_001.pdf (459.47 kb)
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The Skin You Are In: Design-of-Experiments Optimization of Lipid Nanoparticle Self-Amplifying RNA Formulations in Human Skin ExplantsClick to copy article linkArticle link copied!

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Abstract

Results and Discussion

Scheme 1

saRNA Luciferase Expression Kinetics in Human Skin Explants

Figure 1

Effects of Complexing Lipid Identity and Lipid-to-RNA Ratio on Luciferase Expression

Figure 2

Effects of Lipid and Particle Concentration on Luciferase Expression

Figure 3

Effects of Combining Zwitterionic and Cationic Complexing Lipids on Luciferase Expression

Figure 4

Design of Experiments (DoE) Analysis

Figure 5

Figure 6

LNP Delivery and Expression of eGFP saRNA in Human Skin Cells

Figure 7

Identification of Cells Expressing eGFP in Human Skin Cells

Figure 8

Conclusions

Methods

RNA Synthesis and Purification

Production of Lipid Nanoparticles

Particle Characterization

Human Skin Explant Injection, Culture, and Imaging

Flow Cytometry

Design of Experiment and Statistical Analysis

Supporting Information

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Acknowledgments

References

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Abstract

Scheme 1

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

References

Supporting Information

Supporting Information

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The Skin You Are In: Design-of-Experiments Optimization of Lipid Nanoparticle Self-Amplifying RNA Formulations in Human Skin Explants
Click to copy article linkArticle link copied!