Monitoring translation activity of mRNA-loaded nanoparticles in mice

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Monitoring translation activity of mRNA-loaded nanoparticles in mice Sebastian Rosigkeit, Martin Meng, Christian Grunwitz, Patricia Gomes, Andreas Kreft, Nina Hayduk, Rosario Heck, Geethanjali Pickert, Kira Ziegler, Yasmin Abassi, Jasmin Roeder, Leonard Kaps, Fulvia Vascotto, Tim Beissert, Sonja Witzel, Andreas Kuhn, Mustafa Diken, Detlef Schuppan, Ugur Sahin, Heinrich Haas, and Ernesto Bockamp Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00370 • Publication Date (Web): 20 Jul 2018 Downloaded from http://pubs.acs.org on July 22, 2018

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Molecular Pharmaceutics

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Sebastian Rosigkeit1, Martin Meng2, Christian Grunwitz2, Patricia Gomes2, Andreas Kreft3, Nina Hayduk1, Rosario Heck1, Geethanjali Pickert1, Kira Ziegler1, Yasmin Abassi1, Jasmin Röder1, Leonard Kaps1 , Fulvia Vascotto4, Tim Beissrt4, Sonja Witzel4, Andreas Kuhn2, Mustafa Diken2,4, Detlef Schuppan1,5, Ugur Sahin2,4, Heinrich Haas2, & Ernesto Bockamp1

Monitoring translation activity of mRNA-loaded nanoparticles in mice 1

Institute of Translational Immunology (TIM), Medical Center of the Johannes Gutenberg-University, Mainz, Germany 2 BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany 3 Institute of Pathology, Medical Center of the Johannes Gutenberg-University, Mainz, Germany 4 TRON gGmbH, Mainz, Germany 5 Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

Correspondence to: E.B. ([email protected]) and H.H. ([email protected])

Keywords: mRNA nanoparticles, in vivo mRNA delivery, detection method for mRNA nanoparticle delivery

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Abstract Targeting mRNA to eukaryotic cells is an emerging technology for basic research and provides broad applications in cancer immunotherapy, vaccine development, protein replacement and in vivo genome editing. Although a plethora of nanoparticles for efficient mRNA delivery exists, in vivo mRNA targeting to specific organs, tissue compartments and cells remains a major challenge. For this reason, methods for reporting the in vivo targeting specificity of different mRNA nanoparticle formats will be crucial. Here, we describe a straightforward method for monitoring the in vivo targeting efficiency of mRNA-loaded nanoparticles in mice. To achieve accurate mRNA delivery readouts, we loaded lipoplex nanoparticles with Cre recombinase-encoding mRNA and injected these into commonly used Cre reporter mouse strains. Our results show that this approach provides readouts that accurately report the targeting efficacy of mRNA into organs, tissue structures and single cells as a function of the used mRNA delivery system. The here described method establishes a versatile basis for determining in vivo mRNA targeting profiles and can be systematically applied for testing and improving mRNA packaging formats.



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Introduction Combining messenger RNA (mRNA) with nano-sized carriers allows to deliver protein function to eukaryotic cells and will be the basis for a new class of therapeutics1-3. Recent manufacturing advances such as low-cost and large-scale production platforms, the availability of chemically modified mRNA with increased stability and reduced activation of immune sensors and the development of improved nanoparticles now qualify mRNA-based technologies for a wide spectrum of applications1,

4, 5

. In mice, mRNA delivery has been instrumental for preclinical

cancer immunotherapy studies6-9, for testing protective vaccines against infectious agents10-13, for therapeutic protein replacement expeiments14-16 and for in vivo genome editing17-19. First-in-human cancer immunotherapy trials demonstrated therapeutic efficacy for mRNA nanoparticles in melanoma patients20,

21

and human

and veterinary mRNA nanoparticle immunization holds great promise for anti-viral vaccination12, 22. A key prerequisite for such applications is, however, the efficient and on target in vivo delivery of mRNA. Safe and site-specific mRNA delivery will require both profound knowledge of the delivery vehicles23, 24 and information about organs, tissue structures and cell types that are targeted. Thus, methods for monitoring mRNA delivery will be crucial for basic research and for the preclinical evaluation of novel RNA nanomedicines. Here we report a generally applicable method for evaluating mRNA delivery in mice. To record the whole body distribution of mRNA nanoparticle delivery, we first used nanoparticles loaded with firefly luciferase mRNA. In a second step, we injected nanoparticles loaded with Cre recombinase (Cre) mRNA into Cre reporter mice and measured downstream reporter gene activation in organs, tissue sections and specialized cell types. We applied this methodology for further investigating novel, 3 ACS Paragon Plus Environment

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intravenously injectable mRNA nanoparticles, which selectively target specific organs such as spleen and lungs7, 8. These studies provided detailed insight into the targeting characteristics of each tested mRNA nanoparticle. Moreover, we were able to reveal specific delivery patterns inside organs, to analyze the targeting-toexpression ratio and to determine the cell type specificity for different mRNA delivery systems. The here applied strategy facilitates rapid and detailed information about the in vivo delivery of mRNA as a function of the used nanocarrier and thus provides a versatile method for testing and improving mRNA nanoparticle formats in basic research and in the course to clinical translation.



Materials and Methods Mice Luciferase bioluminescence recording was performed with white NMRI mice (TARC, Mainz, Germany). For Cre-recombinase mRNA injection the ROSA26 lacZ Cre reporter (FVB.129S4(B6)-Gt(ROSA)26Sortm1Sor/J)27 or the ROSA26 mT/mG two-color fluorescent Cre reporter (C57/BL6 Gt(ROSA)26Sortm4(ACTB-tdTomato,EGFP)Luo/J)28 mouse strains were purchased from the Jackson Laboratory (Bar Harbor, Maine, USA). In vivo tropism of different mRNA lipoplex formats was tested in mice receiving a single retro-orbital injection consisting of 20 µg mRNA in 200 µl isotonic lipoplex mixture. All mouse experiments were approved by the ethical committee of the Government of Rhineland Palatinate under the reference number 2317707/G15-1-064.

In vitro synthesis of mRNA For producing synthetic mRNAs, firefly luciferase or Cre-recombinase encoding open reading frames were cloned into the T7 polymerase pST1-A30L70 run off vector (TRON gGmbH, Mainz Germany). To generate templates for in vitro transcription, plasmid DNAs were linearized downstream of the poly(A) tail-encoding region using the SapI class II restriction endonuclease, thereby generating a template for transcribing mRNAs lacking additional nucleotides downstream of the poly(A)-tail42. Linearized run off template DNAs were purified, spectrophotometrically quantified and then subjected to in vitro transcription with T7 RNA polymerase as previously described43. The reaction mix contained 7.5 mM each of ATP, CTP, UTP, 1.5 mM GTP and 6 mM beta-S-ACA(D1) cap analog44. After T7 in vitro transcription 5 ACS Paragon Plus Environment

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mRNAs were isolated using magnetic particles45. In each case, the mRNA concentration was determined by spectrophotometry and the integrity of the synthesized mRNA preparation was confirmed using a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA).

Preparation of liposomes and lipoplexes Liposomes and lipoplexes were prepared as outlined in Table 1 and quality controlled at BioNTech RNA Pharmaceutical GmbH (Mainz, Germany). For liposome preparation R-1,2-di-O-oleoyl-3-trimethylammonium propane (chloride salt) (DOTMA, Merck

&

Cie,

Schaffhausen,

Switzerland),

1,2-Dioleoyl-sn-glycero-3-

phosphoethanolamine (DOPE, Avanti Polar Lipids, Alabaster, AL, USA) and cholesterol (CHOL, Sigma-Aldrich, Missouri, MI, USA) was used. NaCl and 99.5% ethanol were purchased from Carl Roth (Karlsruhe, Germany), PBS from Life Technologies GmbH (Darmstadt, Germany) and RNase free water from B. Braun AG (Melsungen, Germany). Summary for generating mRNA lipoplex nanoparticles: Liposome

DOTMA concentration in liposome [mM]

Volume RNA stock 1 mg/ml [µl]

Volume water [µl]

NaCl stock 1.5 M [µl]

Volume liposome [µl]

4.32

Lipoplex charge ratio (+) : (-) 4.0 : 1.0

+DOTMA / DOPE +DOTMA / cholesterol -DOTMA / DOPE -DOTMA / cholesterol

80

418

80

222

2.75

4.0 : 1.0

80

291

80

349

4.32

1.3 : 2.0

80

604

80

36

2.75

1.3 : 2.0

80

583

80

57



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Liposome formation Liposomes were produced by the film hydration method46,

47

. Briefly, stock

solutions of individual lipids were prepared in ethanol at a concentration of 10 mg/ml and the final stock concentrations were confirmed in each case by HPLC. To adjust the intended lipid ratios, appropriate volumes of the lipid stock solutions were mixed and added to a 250 ml round bottom flask. DOTMA/DOPE liposomes were prepared at a 2:1 molar ratio and DOTMA/cholesterol liposomes at a 1:1 molar ratio. Using a rotatory evaporator, the solvent was evaporated and the obtained lipid film was dried for one hour. To obtain a raw colloid with a total lipid concentration of approximately 6 mM, the dry film was hydrated with RNase free water by gently shaking followed by an overnight incubation at 4°C. The resulting dispersion was extruded 10 times through polycarbonate membranes with a 200 nm pore size using the LIPEX® 10 ml extruder (Northern Lipids Inc., Burnaby, Canada). After determining the lipid concentration by HPLC, the final concentration for DOTMA/DOPE liposomes was adjusted to a concentration of 4.3 mM DOTMA and for DOTMA/cholesterol liposomes to a concentration of 2.75 mM DOTMA.

Lipoplex formation Briefly, mRNA was adjusted to a concentration of 1 mg/ml in RNAse free water and mRNA lipoplex nanoparticles were produced by diluting the adjusted mRNA with volumes as outlined in table 1 using RNase free water and 1.5 M sodium chloride followed by the addition of liposome mixes. For the calculation of the molar ratio between RNA and cationic lipid, a mean molar mass of 330 Da per nucleotide and one negative charge per phosphodiesters was assumed. For DOTMA one positive charge per molecule was assumed. 7 ACS Paragon Plus Environment

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Liquid chromatography–mass spectrometry (LC-MS) Sample preparation and extraction For extraction of DOTMA from the biopsies, PBS was added to the organ at equal volume/weigh ratio (e.g. to 100 mg tissue 100 µL PBS were added). Subsequently, samples were disrupted and homogenized using a Tissuelyser (Tissuelyser II Qiagen, Hilden, Germany) applying a frequency of 24 Hz for 10 minutes. From the obtained homogenates, 50 µl were withdrawn and added into 2 ml Eppendorf tubes containing 1.5 ml of a CHCl3:MeOH mixture (1:1, v/v). The tubes were vigorously mixed using a shaker (Multi Reax, Heidolph, Schwabach, Germany) during 40 min, followed by centrifugation at 10.000 rpm for 10 min at room temperature (Heraeus Pico 17, Thermo Fischer Scientific, Waltham, USA). The supernatants were withdrawn and put in 4.5 ml glass vials. This extraction was repeated twice (total of three extractions for each organ) and the extracted volumes were pooled for further analysis.

Liquid chromatography – mass spectrometry (LC-MS) measurements LC-MS analysis was performed on a 1290 Infinity UHPLC consisting of a binary pump connected to a 6460 Triple Quadrupole Mass Spectrometer (Agilent Technologies, Santa Clara, USA) equipped with an electrospray ion source with Jet Stream Technology, which enhances sensitivity and improves robustness. For the liquid chromatographic separation, the reversed phase column used was Zorbax SBC18, RRHD (2.1 x 50 mm, 1.8 µm) (Agilent Technologies, Santa Clara, USA). The column temperature was set at 50°C and the auto sampler temperature was 20°C. 8 ACS Paragon Plus Environment


Injection volume was 1 µl and between each sample injection the needle wash was flushed and washed with methanol/isopropanol 1:1 for 15 seconds. Eluent flow rate was 0.5 ml/min and the eluents were A = 0.1% ammonium acetate/0.1% acetic acid in water/methanol 50/50 and B = 0.1% ammonium acetate/0.1% acetic acid in isopropanol/methanol 50/50. The liquid chromatography gradient was from 50% of A to 100% of B in 3.5 min, where it remained for 4.5 min. The gradient was then decreased from 100% to 50% of B for 30 seconds and the column was stabilized by 1.5 min flow of 50% of A and 50% of B. For the Agilent Triple Quadrupole instrument the electrospray ionization source was operated on positive ion mode and the optimal sources were as following: drying gas temperature 275°C, gas flow 6 l/min, capillary voltage 4500 V, nebulizer gas (nitrogen) pressure 30 psi and the highest parent ion abundance was determined using fragmentor voltages of 280 V. Product ion (MS2) spectra were generated using collision induced dissociation (CID) in the collision cell with nitrogen gas. The most intense product ion signal was achieved by applying offset voltages of 45 eV and 50 eV. Following transitions were selected for multiple reaction monitoring (MRM): m/z 634.5→69.1 (Quantifier) and m/z 634.5→83.1 (Qualifier). Data acquisition and quantitation of the analyte (DOTMA) was performed using the Agilent MassHunter Workstation software.

Particle size measurement Particle size was measured by dynamic light scattering using the DynaPro NanoStar (Wyatt Technology, Dernbach, Germany). From the measurements, average size (ZAverage) polydispersity indices (PI) were calculated from the cumulant analysis using the implemented software. Each measurement was performed with 10 acquisitions per measurements, with 5 seconds per acquisition. 9 ACS Paragon Plus Environment

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Measurements were performed as triplicates (three lipoplex formations from each liposome batch) where for each composition two independently manufactured liposome batches were used. Therefore, for each condition results from overall six measurements were averaged. The statistical error between the individual measurements was calculated.

Zeta potential measurement Zeta potentials were obtained from the electrophoretic mobility, measured with the Wallis ξ- Zeta Potential Analyzer (Corduan Technologies, Pessac, France), using the Smoluchowski formalism. Measurements were performed in 15 mM NaCl solution.

Each

sequence

of

measurements

consisted

of

10

acquisitions.

Measurements were performed as triplicates (three lipoplex formations from each liposome batch) where for each composition two independently manufactured liposome batches were used. The results from all measurements for a given type of sample were averaged and the statistical error between the individual measurements was calculated.

In vivo bioluminescence imaging D/D and D/C formulations were mixed with 20 µg mRNA encoding firefly luciferase (Luc mRNA) and injected into white NMRI mice. Six hours later mice were anesthetized by continuous inhalation with 3% isofluorane. After intraperitoneal injection with 75 mg/kg body weight of D-luciferin (BD Biosciences, Heidelberg, Germany) in PBS and waiting for five minutes to allow distribution of D-luciferin, mice were placed in the chamber of an IVIS Lumina optical imaging system (Caliper Life 10 ACS Paragon Plus Environment


Sciences, Rüsselsheim, Germany) and bioluminescence levels were recorded. The signal intensity was scaled to a pseudocolor image, which was superimposed on a grayscale photo of the mice.

Determination of luciferase activity in protein extracts D/D and D/C formulations were mixed with 20 mg mRNA encoding firefly luciferase (Luc mRNA) and injected into white NMRI mice. Six hours later organs were dissected, rinsed twice in phosphate buffered saline (PBS, pH 7.3) and about half of each biopsy was transferred into a 2 ml safe lock tube and weighted. After adding 2 volumes (w/v) of 1 X Reporter Lysis Buffer (Promega GmbH, Mannheim, Germany) the samples were homogenized using a Qiagen TissueLyzer II (Quiagen, Hilden, Germany), frozen at -20 °C and subsequently centrifuged for 15 minutes at 13000 rpm (4° C) to remove cell debris. Luciferase activity was determined from 10 µg protein with the Luciferase Assay System E4030 (Promega, GmbH, Mannheim, Germany) and measured on an Infinite 200 PRO multimode reader (Tecan, Männedorf, Switzerland).

Flow cytometry ROSA26 mT/mG two-color fluorescent Cre reporter mice were sacrificed 48 h after Cre mRNA injection. Lungs were surgically removed, placed into ice cold petri dishes and minced with scissors. Next, the minced tissue was transferred into 50 ml Falcon tubes containing 6 ml digestion buffer (10 U/ml collagenase, 80 U/ml dispase (both Roche Diagnostics, Mannheim, Germany) in PBS pH 7.3) and incubated at 37° C for 45 minutes. Disaggregated cells were passed through a 70 µm cell strainer 11 ACS Paragon Plus Environment

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(Greiner bio one, Kremsmünster, Austria) and erythrocytes were removed by treating with erythrocyte lysis buffer (174 mM ammonium chloride, 10 mM potassium hydrogencarbonate, 0,1 mM Na2EDTA, pH 7,3) for 90 seconds at room temperature. Lysis was stopped by adding 6 ml DMEM not containing FCS. Cells were centrifuged and washed twice, resuspended in 6 ml PBS and counted. Before antibody staining, cells were diluted to one million cells per 100 µl FCS free PBS and life/dead stained with Fixable Viability Dye eFluor® 455UV (eBioscience, San Diego, USA). Before flow cytometry was performed, one million cells were resuspended in 100 µl FACS buffer (95 % PBS, 5 % FBS) and stained with different antibody mixes (indicated in table 2). Cells acquisition was performed on an LSRFortessa (Becton Dickinson, BD, USA) and analysed with the FlowJo software (TreeStar, Ashland, USA).

List of antibodies:

Antigen CD11c Siglec F MHC II CD103 Ly6C/Ly6G CD11b CD31 F4/80 E Cadherin CD3 CD3 CD45 CD23 (Fcε receptor II) NK 1.1 CD19

Fluorochrome PE-Cy7 BV421 APC BV510 APC-Cy7 PerCp-Cy5.5 PE-Cy7 BV421 BV510 APC-Cy7 BV421 V500 PE-Cy7 PerCP-Cy5.5 PerCp-Cy5.5

Clone

Provider

HL3 E50-2440 AF6-120.1 M290 RB6-8C5 M1/70 390 BM8 DECMA-1 145-2C11 17A2 30-F11 B3B4 PK136 1D3

BD BD BD BD BioLegend BD Abcam BioLegend BD BD BioLegend BD eBioscience BD BD

X-Gal staining and solvent-based tissue clearing Ten days after Cre mRNA injection into ROSA26 LacZ Cre reporter mice, lungs were dissected and fixed in 30 ml cold acetone (stored at -20°C) for 6 h at 4°C. 12 ACS Paragon Plus Environment


For uniform staining buffer penetration, lungs were washed twice with PBS pH 7.3 and equilibrated at 4°C for 6 h in 30 ml equilibration buffer (5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 2 mM magnesiumchloride). To develop β-galactosidase activity, lungs were shifted to X-Gal staining buffer (5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 2 mM magnesium chloride containing 1 mg/ml X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside), incubated for 15 h (if necessary this step can be prolonged up to 48 h) at 37°C and then transferred into ice cold post-fixation buffer (4% formaldehyde/1% glutaraldehyde in PBS pH 7.3). Subsequently, lungs were dehydrated in methanol. For this, lungs were transferred first into 60 ml 25% methanol/75% PBS at room temperature for 1h. This step was repeated with 60 ml of 50% methanol/50% PBS buffer and consecutively with 75% methanol/25% PBS buffer. For dehydration lungs were transferred twice to 60 ml of 100% methanol for at least one hour and then placed into 60 ml of 100 % methanol for 15 h at 4°C. To render the lung tissues transparent, lungs were transferred to a 2:1 mixture of benzyl-benzoate/benzyl-alcohol at room temperature. After clearing whole mount images were generated using a Stemi 2000-C binocular (Carl Zeiss, Oberkochen, Germany) equipped with a SPOT digital microscope camera (Diagnostic Instruments, Sterling Heights, USA) and imported into Photoshop 7.0 (Adobe Systems).

X-Gal stained paraffin sections Lungs of ROSA26 Cre reporter mice injected with Cre mRNA lipoplexes and matched control lungs from mice that did not receive Cre mRNA lipoplexes were acetone-fixed, X-Gal-stained, post-fixed and dehydrated as described in the previous paragraph and subsequently embedded in paraffin. The resulting blocks were 13 ACS Paragon Plus Environment

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sectioned and counter stained with haematoxylin and eosin (H&E). Images were generated using an Axioscope A1 microscope equipped with an AxioCam MRc 5 digital camera (Carl Zeiss, Oberkochen, Germany) and imported into Photoshop 7.0 (Adobe Systems).



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Results Assembly of negatively and positively charged mRNA lipoplexes To gain insight into the organ specificity of mRNA nanoparticle delivery systems, we used lipoplex formulations, which enable site-specific in vivo delivery of mRNA activity by variation of physicochemical properties like charge and molecular composition8. Four different firefly luciferase mRNA (Luc

mRNA) lipoplex

formulations were selected, for which preferential targeting to lung or spleen can be implemented by varying the charge ratio (cationic lipid to RNA)8. Using established protocols25, 26, we assembled these lipoplex formulations by mixing Luc mRNA with two different types of cationic liposomes, both containing the synthetic cationic lipid R-1,2-di-O-oleoyl-3-trimethylammonium propane (DOTMA), and additionally as a helper

lipid,

either

the

zwitterionic

phospholipid

1,2-Dioleoyl-sn-glycero-3-

phosphoethanolamine (DOPE) or cholesterol (CHOL). For both types of liposomes, lipoplexes were assembled at two different lipid to RNA ratios (charge ratios), namely either with an excess of cationic lipid at a charge ratio of 4/1 (+/-) or with an excess of negatively charged mRNA at a charge ratio of 1.3/2 (+/-) (Figure 1). In each case, the charge ratio was calculated as the number of positively charged ammonium groups in the cationic lipid in comparison to the number of negatively charged phosphate groups in the mRNA backbone. Lipoplex nanoparticles with expected size and polydispersity index were obtained where, with an excess of positive charge (4/1) the zeta (ξ) potential was positive while with an excess of negative charge the ξ potential was negative8. Particle sizes and ξ potentials of the lipoplexes are shown in Table 1. The four different lipoplex formulations were then injected into mice (Figure 1).

Organ selectivity of mRNA expression 15 ACS Paragon Plus Environment

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To confirm the expected targeting selectivity for the four different delivery systems, we first provided a general picture about organs and body structures that were targeted. In each case 200 µl of the above-described four different Luc mRNA nanoparticles were injected at an mRNA concentration of 0.1 mg/ml. Six hours later mice were subjected to whole body bioluminescence imaging. Compared to control mice not receiving Luc mRNA nanoparticles, both positively and negatively charged Luc mRNA lipoplexes produced strong bioluminescence signals (Figure 2A). Confirming the previously reported general targeting selectivity of such delivery systems7, 8, cationic formulations primarily targeted the lungs and anionic lipoplexes the spleen (Figure 2A). For quantitative comparison of organ selectivity, we determined luciferase activity in protein extracts from lungs, spleen, heart, liver and kidneys. This analysis revealed that the signal strength was dependent on the helper lipid (Figure 2B). Luciferase expression in the lungs was highest with cholesterol, and in the spleen, highest with DOPE. Notably, there was no detectable activity in liver and other organs, except for a weak signal in the heart for positively charged DOTMA/CHOL lipoplexes (Figure 2B). To correlate mRNA expression with the accumulation of lipid carrier, we extracted DOTMA (the cationic lipid component present in all four formulations) from lungs, spleen, heart, liver and kidney and subsequently measured DOTMA concentrations by liquid chromatography–mass spectrometry (LC-MS). As shown in Figure 2C, DOTMA was found predominantly in lungs, spleen and liver, but was absent from heart and kidneys. However, DOTMA accumulation and Luc mRNA expression did not strictly correspond. This clearly demonstrates that the expression of the mRNA cargo is not necessarily matched by the bioavailability of the lipid carrier. This discrepancy was most notable for the liver where substantial amounts of 16 ACS Paragon Plus Environment


DOTMA accumulated without giving rise to detectable luciferase activity. However, since luciferase expression was measured at six hours after nanoparticle injection, at least, some intact Luc mRNA nanoparticles should have reached, and if possible, delivered their cargo to the liver. This discrepancy between lipoplex nanoparticle accumulation and mRNA protein expression underscores the necessity to control carrier bioavailability and mRNA expression.

Visualizing mRNA delivery in organs and tissues Having demonstrated that positively charged mRNA lipoplex nanoparticles delivered mRNA to the lungs, we next determined the location of targeted cells in this organ. To this end, mRNA encoding Cre-recombinase (Cre mRNA) was synthesized and assembled at a charge ratio of 4/1 (+/-) either with DOTMA/CHOL or alternatively with DOTMA/DOPE. The resulting two mRNA nanoparticle formats were then injected into ROSA26 lacZ Cre reporter mice. These mice harbor a silent lacZ reporter gene in the ROSA26 gene locus that is conditionally activated in cells expressing Cre protein27. To visualize the overall three-dimensional (3D) distribution of Cre mRNA-targeted cells within the lungs, we applied a solvent-based tissue clearing method. Injection of positively charged Cre mRNA lipoplexes into ROSA26 lacZ reporter mice resulted in different lung targeting patterns for DOTMA/DOPE and DOTMA/CHOL formulations. As seen in Figure 3A, lungs of mice injected with DOTMA/DOPE mRNA lipoplexes harbored blue, Cre mRNA-targeted cells in central areas of the left, right cranial, middle and caudal lung lobes. However, no productive reporter gene expression in the right post caval lobe was observed (Figure 3D). The DOTMA/CHOL was more efficient in targeting the lungs than DOTMA/DOPE. This was indicated by the intense proximal and distal X-Gal staining of all five lung lobes 17 ACS Paragon Plus Environment

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(Figure 3B and E). Higher magnification confirmed that DOTMA/DOPE nanoparticles did not deliver Cre mRNA activity to distal airways (Figure 3G). By contrast, DOTMA/CHOL mRNA nanoparticles targeted both proximal and distal airways (Figure 3H). Demonstrating the complete absence of background activity, we observed no X-Gal-stained cells in ROSA26 lacZ Cre reporter mice that were not injected with Cre mRNA (Figure 3C, F and I). To further refine this analysis, X-Gal-stained lung sections were inspected at higher magnification. In line with the above results, ROSA26 lacZ Cre reporter mice receiving DOTMA/DOPE Cre mRNA lipoplexes lacked reporter gene activation in distal lung parenchymal cells, alveolar walls, bronchioles and alveoli (Figure 4A). Conversely, more proximal sections contained patches of lacZ Cre reporter geneexpressing blue cells (Figure 4D). Microscopic analysis also revealed the presence of cells with lacZ activity in distal pulmonary vessels (arrowheads in Figure 4A and 4G). This demonstrated that with the exception of cells contained within pulmonary vessels, DOTMA/DOPE Cre mRNA nanoparticles were principally targeting proximal lungs. Also confirming our previous observations in transparent whole mount lung preparations, DOTMA/CHOL Cre mRNA nanoparticles delivered Cre-recombinase activity to both distal and proximal sites (Figure 4B and E). In addition, we noticed a very uniform staining of endothelial cells in small lung vessels in the DOTMA/CHOL Cre mRNA lipoplex group (arrowhead in Figure 4H). In contrast, both cationic lipoplex formulations failed to deliver Cre mRNA activity to large airway cells (LA in Figure 4G and H). As expected, ROSA26 lacZ Cre reporter lung sections from controls not receiving Cre mRNA did not contain any blue cells (Figure 4C, F and I).

Identification and quantification of targeted cells by flow cytometry 18 ACS Paragon Plus Environment


For many experiments, it will be crucial to accurately assess to what extent a nanocarrier delivers the mRNA cargo to a particular cell type. To identify lungresident cells efficiently targeted by cationic DOTMA/CHOL Cre mRNA nanoparticles, we resorted to a second Cre reporter mouse line. This double-fluorescent Cre reporter mouse constitutively expresses membrane-bound fluorescent tomato protein (mT) and activates membrane-bound green fluorescent protein (mGFP) upon Crerecombinase expression28. Two days after injection of cationic DOTMA/CHOL Luc control or Cre mRNA nanoparticles into tomato/GFP reporter mice, lungs were harvested and analyzed by flow cytometry. As expected, injection with Luc or Cre mRNA lipoplexes did not change the overall numbers of cells expressing the tomato gene (Figure 5A and 5B). Gating on mGFP expression revealed 2.9% mGFP+ cells in lungs receiving Cre mRNA lipoplexes and 0.2% background fluorescence in the Luc mRNA treated controls (Figure 5A and 5B). To obtain detailed information about the identity of mGFP activated cells, we used lineage-specific markers. As shown in Figure 5C, the majority of cells expressing mGFP were endothelial cells (57%), followed by hematopoietic cells (36%) while only 3% of epithelial cells and 4% of not further characterized cell types expressed mGFP. This indicated that the here selected DOTMA/CHOL Cre mRNA formulation primarily targeted lung endothelial and hematopoietic cells. For further refinement, we analyzed the expression by and the distribution within different lungresident cellular subsets. Flow cytometry showed that cationic DOTMA/CHOL Cre mRNA nanoparticles reached about 16% of all epithelial cells, 23% of all endothelial cells and 1% of all CD45+ blood cells (Figure 6A). This means that although 36% of all mGFP-expressing cell in the lungs were blood cells, only about 1% of all lungresident blood cells were efficiently targeted by DOTMA/CHOL Cre mRNA lipoplexes. 19 ACS Paragon Plus Environment

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To analyze hematopoietic subpopulations, we used additional markers. As shown in Figure 6B, very few B, T and natural killer (NK) cells expressed mGFP, indicating that DOTMA/CHOL Cre mRNA nanoparticles did not efficiently target leukocytes. A very different picture emerged when we analyzed macrophages and dendritic cells. Here we found that 12% lung macrophages and 3% lung dendritic cells expressed mGFP (Figure 6C and D). Analysis of lung macrophage subsets revealed that 3% of bone marrow-derived interstitial macrophages29 and 13% of tissue-resident alveolar macrophages30 expressed mGFP. Thus, cationic DOTMA/CHOL Cre mRNA nanoparticles preferentially targeted tissue-resident alveolar macrophages (Figure 6C). Within dendritic cell subsets, 8% of Th1-priming CD11b+ and 6% of Th2-priming CD103+ pulmonary dendritic cells31 expressed mGFP (Figure 6D). From this, we conclude that the here applied method provides a very clear picture about qualitative and quantitative in vivo delivery patterns for any mRNA nanocarrier and that this method will be very suitable for identifying rare and inefficiently targeted cells.

Table 1: Physicochemical properties of anionic and cationic mRNA lipoplex formulations Liposomes Charge ratio +/Z-Average [nm] Polydispersity Index Zeta potential [mV]

DOTMA/DOPE 4.0 : 1.0 (+) 284 ±10 0.20 ± 03

DOTMA/DOPE 1.3 : 2.0 (-) 212 ± 11 0.16 ± 0.03

DOTMA/Chol 4.0 : 1.0 (+) 337 ± 19 0.22 ± 0.02

DOTMA/Chol 1.3 : 2.0 (-) 235 ± 7 0.15 ± 0.03

+9.5 ± 1

-6.0 ± 1.5

+9.5 ± 1

-7 ± 1.5

The size of all nanoparticle products was in a range between 200 and 400 nm, with a good reproducibility between different preparations for a given composition. Positively 20 ACS Paragon Plus Environment


charged lipopexes were significantly larger than those formed having excess negative charge. As expected, lipoplex nanoparticles formed with an excess of negative charge had a negative zeta potential, while those formed with an excess of positive charge had a positive zeta potential.


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Discussion One of the central issues facing the in vivo application of mRNA nanoparticle technology is to control the efficacy and specificity of mRNA delivery. Here, we introduce a straightforward and rapid method for monitoring mRNA nanoparticle delivery in mice. The gold standard for visualizing mRNA delivery in mice has been bioluminescence imaging of luciferase activity or the labelling of mRNA and/or carrier molecules with fluorescent dyes32-34. Furthermore, positron-emission tomography of radiolabeled mRNAs and carriers or the detection of specific mRNA-encoded reporter proteins have been used32. However, the informative value of bioluminescence imaging and positron-emission tomography is limited because both methods will not generate high-resolution images or detect single cells. Only when combined with more sophisticated methods such as photoacoustic tomography serial block-face imaging35,

36

or optical-sectioning microscopy37, high-resolution 3D images can be

produced. The here introduced method, however, does not rely on sophisticated equipment and our studies demonstrate the method to be highly efficient and accurate. Moreover, for monitoring mRNA nanoparticle delivery, commonly available Cre reporter mouse strains containing different reporter genes such as lacZ, fluorescent proteins or luciferase may be used27,

38-41

. By choosing a specific Cre

reporter strain, readouts thus can be specifically adapted to the particular requirements of the experiment or the available detection methods. To facilitate 3D imaging of mRNA-targeted cells within organs, we developed a fast solvent-based tissue clearing method. When combined with a detection method that visualizes mRNA-targeted cells, whole mount images showing the spatial distribution of targeted cells are generated. Here we have used X-Gal staining/tissue 22 ACS Paragon Plus Environment


clearing to investigate the distribution of cationic mRNA lipoplex delivery in lungs of ROSA26 lacZ Cre reporter mice. Interestingly, we found that DOTMA/DOPE and DOTMA/CHOL mRNA nanoparticles generated different lung targeting patterns. Subsequent microscopic analysis of X-Gal-stained tissue sections confirmed the different zonal compartmentalization of targeted cells and allowed to visualize targeted cell types within the tissue architecture of the lungs at single cell resolution. To avoid unwanted off targeting and for accurate interpretation of in vivo mRNA lipoplex nanoparticle applications, it will be important to monitor the identity of targeted cell types and to quantify their numbers. To test if Cre mRNA-loaded nanoparticles are suited for this purpose, we injected cationic DOTMA/CHOL Cre mRNA lipoplexes into tomato/GFP Cre reporter mice. Flow cytometry of lung cells provided the necessary information about cell types that were efficiently targeted and allowed to quantify the overall percentages of cells expressing the mRNA cargo. For example, analysis of lung-resident hematopoietic cells demonstrated that cationic DOTMA/CHOL Cre mRNA lipoplexes failed to efficiently target leukocytes (B, T and NK cells) but were able to deliver mRNA activity to dendritic cells and macrophages. Application of additional lineage markers also facilitated the analysis of rare and highly specialized blood cell types such as alveolar and interstitial macrophages or Th1- and Th2-priming lung dendritic cells. Given the speed, simplicity and flexibility of the described method, we anticipate that this approach will prove very valuable for studying mRNA nanoparticles in mice. Moreover, because loading of nanocarriers with Cre mRNA provides accurate information about organ, tissue and cell type-specific delivery, this methodology is likely to become a standard technique in experimental mouse models


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and for stratifying the development of novel mRNA therapeutics for future clinical applications.

Acknowledgements We

thank

Tijana

Bacic

and

André

Gerths

from

BioNTech

RNA

Pharmaceuticals GmbH for dynamic light scattering measurements to determine size and zeta potential of the lipoplex formulations. We are also grateful to our animal technicians. This work was supported by a grant from the Stiftung Rheinland-Pfalz für Innovation (S.R. and E.B.).



Figure legends Figure 1: Principle of mRNA lipoplex nanoparticle generation Negatively charged synthetic mRNA and positively charged liposomes used for assembly of the lipoplex nanoparticles are shown on the left. Lipoplex formulations were assembled either with a 1:4 charge ratio between liposomes and mRNA (cationic lipoplex) or with a 1.3:2 charge ratio of liposomes to mRNA (anionic lipoplex) and are depicted in the centre. In vivo delivery potentials of the different mRNA lipoplexes were tested by injecting the assembled cationic and anionic nanoparticles into mice.

Figure 2: Cationic lipoplexes target mRNA activity preferentially to the lungs (A) Whole-body and organ bioluminescence imaging of non-injected control mice or animals that were treated with anionic or cationic Luc mRNA lipoplex nanoparticles. A representative whole-body bioluminescence image is shown together with the bioluminescence analysis of heart, lungs, liver, spleen and kidneys (from left to right). To visualize luciferase activity, the measured photon counts were translated into pseudo-colours. (B) Determination of luciferase activity in protein extracts from lungs, spleen, heart, liver and kidneys. (C) DOTMA-detecting liquid chromatography–mass spectrometry (LC-MS) analysis of lungs, spleen heart, liver and kidneys. In B and C the different lipoplex formulations are indicated below each bar and a minimum of five animals was used for each experiment. +D/C (cationic 4:1 DOTMA/CHOL lipoplexes); -D/C (anionic 1.3:2 DOTMA/CHOL lipoplexes); +D/D (cationic 4:1 DOTMA/DOPE lipoplexes); -D/D (anionic 1.3:2 DOTMA/DOPE lipoplexes).


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Figure 3: Cationic +D/D and +D/C mRNA lipoplex nanoparticles deliver Crerecombinase activity to different parts of the lung Images A-C show all five lung lobes as transparent whole mounts that were obtained from ROSA26 lacZ Cre reporter mice (A) ten days after a single injection with +D/D Cre RNA lipoplexes (B) ten days after a single injection with +D/C Cre mRNA lipoplexes and (C) without Cre mRNA injection. In the middle panel lung post caval lobes from (D) a +D/D Cre mRNA-injected, (E) a +D/C Cre mRNA-injected and (F) a ROSA26 lacZ Cre reporter mouse that was not Cre mRNA injected are depicted. Images in the lower panel show a higher magnification of the upper left lung lobe from (G) a +D/D Cre mRNA-injected, (H) a +D/C Cre mRNA-injected and (I) a not injected ROSA26 lacZ Cre reporter mouse. Lung lobes shown from top down in A-C are right-lung cranial lobe, right-lung post caval lobe, left-lung lobe, right-lung middle lobe and right-lung caudal lobe.

Figure 4: X-Gal staining reveals differential location of +D/D and +D/C Cre mRNA-targeted cells within distal and proximal lung areas Images A-C show representative distal lung sections from ROSA26 lacZ Cre reporter mice (A) ten days after a single injection with +D/D and (B) ten days after a single injection with +D/C Cre mRNA lipoplexes and (C) without Cre mRNA treatment. Images D-F show proximal lung sections from central lung areas of ROSA26 lacZ Cre reporter mice (D) ten days after a single injection with +D/D, (E) ten days after a single injection with +D/C Cre mRNA nanoparticles and (F) without Cre mRNA treatment. In the lower G-I panels higher magnifications depicting large airways (LA) and lung vessels (V) from ROSA26 lacZ Cre reporter mice (G) ten days after a single injection with +D/D, (H) ten days after a single injection with +D/C Cre mRNA 26 ACS Paragon Plus Environment


nanoparticles and (I) without Cre mRNA treatment are depicted. Arrowheads indicate X-Gal stained cells within the pulmonary vessels.

Figure 5: Percentages and distribution of cells targeted by +DC Cre mRNA lipoplex nanoparticles (A) Representative FACS plots of mT/mGFP Cre reporter mouse lung cells that had been injected once with +DC control Luc mRNA (upper plots) or with +DC Cre mRNA nanoparticles (lower plots). The percentages of fluorescent mT+ and mGFP+ lung cells are indicated in the upper right corner of each plot. (B) Mean percentages and standard deviations of mT+ (red bars) and mGFP+ (green bars) fluorescent lung cells in mT/mGFP Cre reporter mice that have been injected with Luc or Cre mRNA. Each bar represents the mean values of GFP+ cells. (C) Percentages of different lung cell types that were targeted by the +DC Cre mRNA nanoparticles. A minimum of five animals were analysed in each experiment.

Figure 6: Percentages of cells targeted within selected non-hematopoietic and hematopoietic cell types Pie charts on the left indicate the percentages of mGFP+ epithelial, endothelial and blood cells in the lung that had been efficiently targeted after a single injection with +DC Cre mRNA nanoparticles. Central pie charts show the mean percentages of mGFP+ B, T and natural killer (NK) cells. Pie charts on the right show the mean percentages of mGFP+ macrophage and dendritic lung cell populations and their more specialized subsets. Analyzed cell types and specific markers used for


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identifying each cell population are indicated above each pie chart. A minimum of five individual mice was used for each analysis.



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250x138mm (300 x 300 DPI)


Monitoring translation activity of mRNA-loaded nanoparticles in mice

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