A nature-inspired nanodelivery platform for gene silencing in hematopoietic stem and progenitor cells

doi:10.21203/rs.3.rs-3458536/v1

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A nature-inspired nanodelivery platform for gene silencing in hematopoietic stem and progenitor cells

https://doi.org/10.21203/rs.3.rs-3458536/v1

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Nucleic acid therapeutics harbor great potential for silencing, expressing, or editing genes. Here, we introduce a nanodelivery platform based on natural lipoproteins, which prevents premature degradation of small interfering RNA (siRNA), ensuring its targeted and intracellular delivery to hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. After establishing a prototype apolipoprotein lipid nanoparticle (aNP) that stably incorporates siRNA in its core, we built a comprehensive library of which we thoroughly characterized the individual aNPs’ physicochemical properties. Following the in vitro screening of all formulations, we selected eight siRNA-aNPs that are representative of the library’s diversity, and determined their capacity to silence lysosomal-associated membrane protein 1 (LAMP1) in immune cell subsets in mice, using an intravenous administration regimen. Our data show that using different aNPs, we can achieve functional gene silencing in immune cell subsets and their bone marrow progenitors. Beyond gene silencing, the aNP platform’s inherent capacity to engage immune cells provides it with considerable potential to deliver other types of nucleic acid therapeutics to HSPCs.

Biological sciences/Biotechnology/Nanobiotechnology/Nanoparticles

Biological sciences/Biotechnology/Biomaterials/Drug delivery

Genetic drugs’ real-life potential is critically dependent on delivery technology that stabilizes the nucleic acid payload and prevents its premature clearance and degradation, while actively delivering the payload to the cells of interest¹. In the past decade, lipid nanoparticle (LNP) technology emerged that enabled the first small interfering RNA (siRNA) therapeutic’s clinical translation, involving gene silencing in hepatocytes for the treatment of hereditary transthyretin amyloidosis^2,3. Further, LNP technology was essential for the COVID-19 messenger RNA (mRNA) vaccines’ development and successful deployment^4,5. While for most vaccination purposes intramuscular administration is adequate, following intravenous administration, LNPs preferentially accumulate in the liver’s hepatocytes⁶. To enable targeting of tissues and cells beyond the liver, several LNP screening and modification strategies have been developed and evaluated preclinically. These include incorporating charged phospholipids^7,8 in, and antibody surface modification of LNPs^9–14, as well as DNA barcoding strategies^15,16. However, to unlock RNA therapeutics’ full potential for systemic delivery, novel technologies must be developed that possess superb biocompatibility profiles and tunable biodistribution features.

Here, we introduce a nanodelivery strategy based on lipoprotein trafficking. Lipoproteins are endogenous, nanosized transport systems composed of apolipoproteins and aggregates of fatty molecules¹⁷. They inherently interact with a variety of cells and exhibit compelling features for RNA delivery¹⁸. The widely studied apolipoprotein A1 (apoA1) is high-density lipoprotein’s (HDL) main protein constituent¹⁹. Capitalizing on apoA1’s natural function, we developed designer nanotechnology^20,21 to deliver siRNA to myeloid cells and hematopoietic stem and progenitor cells (HSPCs) in the bone marrow^22–24 (Fig. 1a). After establishing a prototype apolipoprotein lipid nanoparticle (aNP) that stably incorporates siRNA in its core, we built a comprehensive siRNA-aNP library and physicochemically characterized and screened individual formulations’ in vitro properties. We subsequently selected eight siRNA-aNPs that are representative of the library’s diversity, and in vivo screened their capacity to silence lysosomal-associated membrane protein 1 (LAMP1) in diverse immune cell subsets in mice. Finally, we studied the lead siRNA-aNP candidate’s physicochemical properties, tissue biodistribution, and in vivo silencing in HSPCs.

Prototyping apolipoprotein lipid nanoparticles for siRNA immune cell delivery

First, we developed a two-step flow manufacturing process for an aNP prototype containing siRNA, the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), the ionizable lipid (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl-4-(dimethylamino)butanoate (MC3), tricaprylin, cholesterol, and apoA1. Our methodology entails producing a siRNA-lipid assembly intermediate through rapid mixing of the components at pH 4 using a T-junction mixer, followed by dialysis against PBS at pH 7.4, and forming stable nanoparticles through apoA1 incorporation using a second rapid mixing step (Supplementary Fig. 1). The prototype aNP input components depicted in Fig. 1b consist of 46 wt% tricaprylin, a triglyceride that makes up the core matrix for MC3-complexed siRNA, DMPC and cholesterol, and 5 wt% apoA1. Importantly, we found siRNA recovery, entrapment, and retention to be higher than 80% (Fig. 1c), indicative of efficient and stable siRNA incorporation. The prototype aNP contained consistent quantities of apoA1 (Fig. 1d) and had a mean hydrodynamic diameter of approximately 80 nm (Fig. 1e), with a favorably low dispersity of 0.1, as measured by dynamic light scattering (DLS). We qualitatively corroborated both size and size distribution by cryogenic electron microscopy (cryo-EM, Fig. 1f), which revealed spherical core-shell structures. Using a gold nanoparticle antibody labeling method, we visualized apoA1’s integration in the aNP by negative staining transmission electron microscopy (TEM, Fig. 1g).

In an in vitro silencing assay using RAW 264.7 murine macrophages that stably express firefly- and renilla luciferase, we found the IC50 value of our aNP prototype to be 14.5 nM (Fig. 1h), which was considerably better than 154.3 nM for an MC3-based control lipid nanoparticle (LNP) formulation. Encouraged by these findings, we developed a unique siRNA radiolabeling strategy (Fig. 1i and Supplementary Fig. 2) to quantitively measure siRNA biodistribution by in vivo positron emission tomography (PET) and ex vivo gamma counting. Using azide-alkyne click chemistry, we functionalized siRNA with desferrioxamine B (DFO) to enable radiolabeling with zirconium-89 (⁸⁹Zr, Fig. 1i and Supplementary Fig. 2). While DLS disclosed a slightly increased hydrodynamic diameter of approximately 95 nm with a uniform size distribution (Fig. 1j), cryo-EM (Fig. 1k) showed the same spherical core-shell structure as the nonlabelled aNP (Fig. 1f). In addition to bare ⁸⁹Zr-labeled siRNA (⁸⁹Zr-siRNA), we intravenously administered ⁸⁹Zr-siRNA-LNP and ⁸⁹Zr-siRNA-aNP to C57BL/6 mice (Fig. 1l, m, at a dose of 8 µCi/mouse, n = 3–5 mice/group). At 24 hours, we subjected mice to in vivo PET imaging, after which they were sacrificed for ex vivo gamma counting of ⁸⁹Zr in various tissues and organs. PET images displayed clear differences between bare ⁸⁹Zr-siRNA’s biodistribution (Fig. 1l), showing renal clearance, and LNP or aNP formulated ⁸⁹Zr-siRNA. Importantly, PET imaging revealed enhanced ⁸⁹Zr-siRNA uptake in the spleen and bone marrow for aNP as compared to LNP delivery. Indeed, quantitively measuring ⁸⁹Zr-siRNA using gamma counting (Fig. 1m) corroborated our in vivo PET imaging findings. Bare ⁸⁹Zr-siRNA was primarily found in the kidneys, while aNPs delivered ⁸⁹Zr-siRNA to the bone marrow (5.5-fold, p < 0.0001) and spleen (2.5-fold, p < 0.0001) significantly better than control LNPs.

After observing the favorable accumulation in hematopoietic organs, we evaluated the prototype aNP’s silencing potential in leukocytes. Towards that goal, we first ex vivo screened eight siRNA constructs against the lysosomal-associated membrane protein 1 (LAMP1, Supplementary Fig. 3). LAMP1 is a compelling target for screening purposes as it is expressed on the cell surface and therefore can be quantified by flow cytometry, as has been demonstrated by Da Silva Sanchez et al¹⁶. We subsequently formulated the most potent anti-LAMP1 siRNA (siRNA_LAMP1) into the prototype aNP formulation. In a proof-of-concept in vivo study, we administered four intravenous siRNA_LAMP1-aNP doses (0.5 mg/kg siRNA_LAMP1, injected 36 hours apart) to six C57/BL6 mice. As compared to an aNP that incorporates scrambled siRNA, we observed significant LAMP1 knockdown in leukocytes in the spleen and bone marrow, which is mostly attributed to LAMP1 silencing in myeloid cells (Fig. 1n). The in vivo siRNA_LAMP1-aNP silencing potency was on par with the positive control siRNA_LAMP1-LNP. Encouraged by our prototype siRNA-aNP’s in vivo performance, we set out to optimize the delivery platform through aNP library screening.

Establishing and characterizing an apolipoprotein lipid nanoparticle library

After prototyping the siRNA-aNP delivery concept, we designed an aNP optimization strategy, involving an iterative design process that ultimately resulted in a library of 72 compositionally distinct (Supplementary Table 1) and fully characterized siRNA-aNPs. To achieve this, we formulated and trialed more than 300 aNPs. Within the library of siRNA-aNP formulations, we kept siRNA and apoA1 quantity constant while varying the cholesterol, tricaprylin, and ionizable lipid MC3 content (Fig. 2a, b). MC3 is a lipid that complexes negatively charged siRNA to allow its integration into an aNP’s lipophilic core, cholesterol and tricaprylin provide structural features that are essential for an aNP’s supramolecular organization and structural stability. Amphiphilic phospholipids are required to disperse the lipophilic supramolecular nanoaggregates of siRNA, MC3, tricaprylin, and cholesterol in an aqueous environment and together with apoA1 stabilize the aNP. We selected 3 phospholipids that are known to properly interact with apoA1²⁵, namely DMPC, 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). After formulating more than two dozen DPPC-based aNPs, we did not identify stable formulations with adequate in vitro silencing features (Supplementary Fig. 4) and decided to exclude these formulations from our library and further testing.

The different aNPs’ compositions and quality features are schematically depicted in Fig. 2c. Although we observed increased tricaprylin to consistently improve DMPC-based formulations, this did not apply to aNPs containing POPC. In general, cryo-EM (Fig. 2d) revealed that the inclusion of tricaprylin promoted the formation of spherical structures, but we also found other morphologies such as aNP₃₉. In the absence of tricaprylin (aNP_{1 to 24}), cryo-EM revealed an array of different morphologies, ranging from spherical (e.g., aNP_{4, 7, 10, 11}), to multilamellar for some POPC-based formulations (e.g., aNP_{3, 6, 15}), and elliptical (e.g., aNP₃₀), but also shapes that were less well defined, such as aNP₁₈ and aNP₂₁. In Supplementary Fig. 5, a detailed overview of cryo-EM images and size analyses (Supplementary Table 2) is provided.

As a critical quality feature, we measured aggregation of individual aNPs directly after production by semi-quantitative assessment of cryo-EM images (Supplementary Fig. 6) and found 55 formulations to pass our predefined threshold of < 15%. Additionally, we determined the aNPs’ stability at 4 °C by DLS. Specifically, we measured size dispersity over a period of 28 days. Interestingly, we observed little correlation between initial aggregation (Fig. 2e) and shelf-life stability (Fig. 2f). Finally, we measured all 72 aNPs’ gene silencing capacity in vitro (Fig. 2g). Similar to other aNP features, we observed diverse gene knockdown capabilities at a fixed concentration of 100 nM siRNA, ranging from no silencing to nearly complete gene silencing. In vitro silencing capacity is a poor predictor of in vivo silencing following intravenous administration²⁶. Therefore, we primarily used this silencing metric as a quality control. Overall, the DMPC-based aNPs were prone to aggregate less but did not appear to be more stable. By setting the quality control limits to 15 %for aggregation, a maximum of 40 %increase in PDI at 28 days, and at least 30 %silencing, we identified 30 aNPs with potentially favorable in vivo features. From those 30 formulations, we selected eight representative aNPs (Fig. 2c, in bold) to be included in ensuing in vivo gene silencing experiments in mice. Our selection was based on identifying aNPs that are representative of the library’s diversity. We included three POPC-based formulations, one of which did not contain tricaprylin, and five DMPC-based formulations, with varying quantities of tricaprylin. Throughout this selection of eight aNPs, the formulations’ morphologies, compositions and nitrogen-to-phosphate ratios vary.

Functionally screening apolipoprotein lipid nanoparticles in vivo

Following aNP library characterization and in vitro screening, we selected eight representative aNPs for in vivo testing in mice, including the aNP prototype (aNP₇₂). Similar to the experiments we conducted with the aNP prototype, we encapsulated siRNA_LAMP1 in the selected aNPs. We assessed these eight siRNA_LAMP1-aNPs’ knockdown functionality using bone marrow-derived macrophages (BMDM) from mice ex vivo. At 100 nM siRNA_LAMP1, reverse transcription quantitative polymerase chain reaction (RT-qPCR) measurements disclosed that all eight aNPs substantially reduced LAMP1 expression (Fig. 3a). After establishing their functionality, we intravenously administered the eight different siRNA_LAMP1-aNPs four times, with 36-hour intervals at 0.5 mg/kg siRNA_LAMP1, in groups of four to six mice per formulation (Fig. 3b). Following this siRNA_LAMP1-aNP treatment regimen, we sacrificed the mice and collected bone marrow cells. These cells were stained with flow cytometry antibody panels that we optimized for the detection of hematopoietic stem cells, myeloid progenitors, and mature myeloid cells (Fig. 3c and Supplementary Fig. 7). Within the aNP selection, we found a remarkable diversity in LAMP1 gene silencing features in immune cells (Fig. 3d, e and Supplementary Fig. 8, 9, 10). Foremost, we observed significant LAMP1 silencing in hematopoietic stem cells using aNP₈, aNP₁₈ and aNP₆₇. While aNP₈ and aNP₁₈ were also capable of significantly silencing LAMP1 in myeloid progenitor cells, we did not observe significant LAMP1 silencing in this progenitor subset using aNP₆₇. We did, however, measure significant LAMP1 silencing in myeloid progenitors using aNP₇₂, a formulation that did not exert any effects on hematopoietic stem cells. A comprehensive overview of the different siRNA_LAMP1-aNPs’ in vivo silencing features is shown in Fig. 3f. It discloses aNP₈’s and aNP₁₈’s potential for broad gene silencing of stem and progenitor populations, while aNP₆₇ silencing displays a bias towards stem cells. For gene silencing specifically in myeloid progenitor cells, aNP₇₂ is most suitable. In light of aNP₁₈’s broad silencing features, we selected this formulation for further studies that focused on reproducibility, pharmacokinetics, safety and pharmacodynamics.

In-depth profiling of lead siRNA apolipoprotein lipid nanoparticle

Starting with the siRNA-aNP prototype (Fig. 1), following comprehensive library screening, and identifying broad gene silencing features (Fig. 2, 3), we selected aNP₁₈ for an in-depth analysis. We incorporated siRNA in aNP₁₈, a DMPC-based formulation that does not contain tricaprylin (Fig. 4a). As holds true for all the formulations that met the predefined quality thresholds, siRNA-aNP₁₈ has siRNA recovery, entrapment, and retention values of more than 80% (Fig. 4b), with consistent apoA1 quantities (Fig. 4c), for multiple batches. In the absence of tricaprylin, the aNP’s hydrodynamic diameter was approximately 50 nm, with a narrow size distribution (PDI < 0.2, Fig. 4d). Encouraged by these strong reproducibility data, we subjected six independently produced siRNA-aNP₁₈ batches to cryo-EM. Cryo-EM disclosed the preparations to predominantly consist of multilamellar, elliptical structures and this was consistent across the six aNP₁₈ batches that we analyzed (Fig. 4e).

After establishing satisfactory reproducibility, we studied the in vivo features of a single siRNA-aNP₁₈ dose in mice. Using the same method that we applied for the prototype aNP, we radiolabeled a siRNA with ⁸⁹Zr, followed by integration into aNP₁₈ (Fig. 1i). 24 hours following intravenous administration into nine mice (at a dose of 8 µCi/mouse), we sacrificed the mice and subjected different tissues to gamma counting (Fig. 4f). For this specific aNP formulation, we observed a relatively strong bone marrow accumulation and an approximately 3-fold higher uptake in the spleen. As many myeloid cells, including myeloid progenitor cells, migrate from the bone marrow to the spleen, we deem the high splenic uptake to be beneficial. The comparably high liver uptake, which we found to be lower than for control LNPs (Fig. 1m), and kidney accumulation, must be taken into consideration and carefully monitored. We therefore measured the liver enzymes alanine transaminase (ALAT) and aspartate aminotransferase (ASAT), as well as the kidney toxicity markers creatinine and blood urea nitrogen (BUN) (Fig. 4g). In line with the liver and kidney toxicity data, ELISA did not uncover immune activation following a single siRNA-aNP administration (at a dose of 0.5 mg/kg) (Fig. 4h). We measured low tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) concentrations comparable to those observed in mice that received intravenous PBS, and significantly lower than the TNFα and IL-6 concentrations following intraperitoneal LPS (0.5 mg/kg) administration, used as positive control.

Finally, we evaluated aNP₁₈’s potential for functional gene silencing of Src homology region 2 domain-containing phosphatase-2 (SHP2). SHP2 is a widely studied therapeutic target in oncology, for example in metastatic breast cancer²⁷. Relevant to the current study and using conditional knockout mouse models, Christofides et al. implicated SHP2 in myeloid cell-driven immunosuppression of the tumor microenvironment²⁸. We established an aNP₁₈ formulation containing siRNA against SHP2, which we refer to as siRNA_SHP2-aNP₁₈. We first evaluated siRNA_SHP2-aNP₁₈’s functionality on murine bone marrow-derived macrophages ex vivo and found dose-dependent silencing effects (Fig. 4i). Finally, we conducted an in vivo silencing study and applied siRNA_SHP2-aNP₁₈ intravenously in C57/BL6 mice (n = 5 per group) inoculated with B16F10 cells according to the regimen depicted in Fig. 4j. Although SHP2 gene silencing varied between individual mice, RT-qPCR disclosed a trend towards a 3-fold reduction in SHP2 mRNA content (Fig. 4k), indicative of the aNP platform’s therapeutic potential.

Lipid molecules, such as cholesterol and tricaprylin, are typically poorly soluble in water and transported throughout the body via endogenous nanoparticles known as lipoproteins¹⁷. For example, reverse cholesterol transport is facilitated by HDL, which is synthesized in the intestines and liver to carry fatty molecules from peripheral tissues back to the liver. HDL can accept cholesterol from myeloid cells, such as macrophages in inflammatory tissues, a process that is mediated by passive diffusion, and via ATP-binding cassette transporters and scavenger receptors²⁹. In addition to interacting with mature myeloid cells, HDL has been shown to directly interact with HSPCs³⁰. While lipoproteins fulfill many important tasks in lipid metabolism^31,32, HDL has also been implicated in transporting RNAs in the human body¹⁸. Owing to these diverse features, HDL and its trafficking in the human body provide a compelling framework for RNA delivery to immune cells and their progenitors in the bone marrow. Although reconstituted HDL or HDL-mimicking nanoparticles have been developed for delivering cholesterol-conjugated siRNA, this strategy results in hydrophilic siRNA’s surface-integration through the lipophilic cholesterol anchor that is incorporated in the phospholipid layer³³. This approach has been explored for hepatic or tumor delivery via scavenger receptor type B1 (SR-B1)^34,35 and vaccine purposes^36,37. However, to efficiently target myeloid (progenitor) and other immune cells, and deliver RNA to their cytosol, RNA must be efficiently and stably incorporated into the HDL core. To accomplish this, we here presented the development and evaluation of the aNP platform. In addition to the platform’s versatility and reproducibility, we have also focused on its diversity in the current study. By varying the aNP composition (Supplementary Table 1), we can finetune properties to optimize RNA delivery to a variety of myeloid cells, as well as HSPCs in the bone marrow. Although LNPs have been shown to reach bone marrow endothelial cells^38–40, antibody conjugation is generally required for efficient HSPC targeting^41,42. One should take into account that LNP technology was not specifically designed for immune cell targeting, but can be adjusted for it through surface modification with targeting moieties. Conversely, we specifically designed the aNP platform with immune cell and HSPC targeting in mind and achieved gene silencing in these cell subsets. In addition to silencing with siRNA, aNPs are suitable for the cytosolic delivery of messenger RNA (mRNA) and antisense oligonucleotides (ASOs). We foresee aNP delivery technology to be further developed as a designer platform for therapeutic immunoregulation in a wide range of immune-mediated diseases, for diverse vaccination applications, as well as for gene HSPC editing in certain hereditary diseases.

In conclusion, we developed the aNP platform for efficient and safe delivery of siRNA to achieve gene silencing in myeloid cells and their progenitors. The aNP platform’s biocompatibility and modularity facilitate systemic administration of siRNA for diverse applications, ranging from silencing genes in HSPCs in the bone marrow to circulating myeloid cells and monocyte-derived macrophages. As shown in this study, using different aNPs, functional gene silencing in different immune cell subsets can be achieved. Beyond gene silencing, the platform has considerable potential for immune cell delivery of other types of nucleic acids, including ASOs and mRNA.

Materials

POPC (Avanti Polar Lipids, 850457)
DMPC (Avanti Polar Lipids, 850345)
DSPC (Avanti Polar Lipids, 850365)
Cholesterol (Sigma)
Tricaprylin (Sigma)
PEG(2K)-DMG (Avanti Polar Lipids, 880151)
DLin-MC3-DMA (SymoChem)
Sodium acetate (Sigma)
12–14 kDa MWCO Dialysis membrane, Spectra/Por™
Quant-iT RiboGreen reagent (ThermoFisher)
TE buffer (10 mM Tris-HCl, 1 mM, EDTA, pH 7.5 in DEPC-treated water)
Apolipoprotein A1 FS assay (DiaSYS)®
Spin filters 100,000 Da MWCO (Amicon)
LabAssay Phospholipid (Fujifilm, Wako)
Cholesterol FS assay (Diasys)
Dual-Luciferase® Reporter Assay System (Promega)
HDL plasma fraction (Lee Biosolution)
RAW264.7 macrophages
Lipofectamine RNAiMAX (ThermoFisher)
Deferoxamine-DBCO (Macrocyclics)
2 kDa MWCO Dialysis tubing (Sigma-Aldrich)
Corning® 70 µm cell strainer (Merck)
CD11b MicroBeads (Miltenyi Biotec)

siRNA-aNP production

siRNA-loaded aNPs were formulated by rapid T-junction mixing. The lipid molecules (POPC, DMPC or DSPC, cholesterol, tricaprylin, (PEG-DMG for LNP particles), and DLin-MC3-DMA) were, in specified ratios (Supplementary Table 1), dissolved in ethanol. siRNA was dissolved in 25 mM sodium acetate (pH 4). Next, organic and aqueous phases were mixed through a T-junction at a flow rate ratio of 1:3 (organic-to-aqueous) and directly collected in a 12–14 kDa MWCO dialysis membrane (Spectra/Por™). Nanoparticle formulations were dialyzed against 1x PBS (pH 7.4) for 4 hours and refreshed for dialysis overnight at 4°C and stirred at 150 rpm. Excess organic and aqueous phases were stored at 4°C to be used for the physicochemical analysis. On the subsequent day, samples were collected from the dialysis bags and the volume was determined. For the aNP particles, the appropriate amount of Apolipoprotein A1 in PBS was diluted to 1/3rd of the sample volume. The apolipoprotein was introduced to the bare formulations by rapid T-junction mixing at a flow rate ratio of 1:3 (apolipoprotein solution/nanoparticle solution). After addition of apolipoprotein, samples were incubated at room temperature for one hour. siRNA-aNPs were filtered through a 0.2 µm filter and concentrated and purified by centrifugal filtration in a 100,000 MWCO filter. Samples were diluted to the desired siRNA concentration and stored at 4°C until further use. Aseptic techniques were used after 0.2 µm filtration.

siRNA-aNP physicochemical analysis

The hydrodynamic diameter of formulated particles was expressed as the number-weighted mean diameter provided by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments, Worcestshire, UK) equipped with a Malvern Zetasizer NanoSampler (Malvern Instruments, Worcestshire, UK). The size dispersity was measured as the polydispersity index (PDI). For the DLS measurements, 100 µL of the formulation sample was diluted into 700 µL of 1X PBS and equilibrated at room temperature before the analysis. Each sample was measured 5 times, 10 runs of each 10 seconds, without fixing the attenuator and measurement position. The Quant-iT RiboGreen assay (Thermo Fisher) was used to quantify the amount of siRNA loaded inside formulated particles. A standard formulation sample with a theoretical siRNA concentration of 133.3 µg/mL, was diluted 200 times in TE buffer and TE buffer containing 2% Triton™ X-100, in a black 96-well plate to a total volume of 100 µL. The Triton detergent disrupts the lipid-based nanoparticles; therefore, total siRNA (retained and unretained) becomes accessible for the Quant-iT RiboGreen reagent. Known controls of siRNA, stored during formulation, were diluted 53.3 times in both TE buffer and TE buffer containing 2% Triton to a total volume of 100 µL. Next, RiboGreen reagent was diluted 200 times with TE buffer and TE buffer containing Triton X-100. 100 µL of this dilution was added to each well containing sample or control to bring the total volume to 200 µL. Subsequently, the samples’ fluorescence was measured on a Tecan Spark® microplate reader at an excitation wavelength of 480 nm and emission wavelength of 520 nm. Recovery of siRNA was determined as (amount of siRNA (retained + unretained))/(total amount of siRNA (used in the formulation)) x 100%, and the entrapment of siRNA was defined as (1-((amount of siRNA (retained + unretained))/(total amount of siRNA (used in the formulation)))) x 100%. The siRNA retention = recovery x entrapment. Cholesterol was quantified using the Cholesterol FS assays (DiaSYS), an enzymatic colorimetric test. 240 µL of buffer containing color reagent was added to 10 µL of sample in a transparent 96-well plate and incubated at 37°C for 5 minutes. The absorbance was measured at 500 nm using a Tecan Spark® plate reader. Phospholipid was quantified using the LabAssay(™) Phospholipid (FUJIFILM), an enzymatic colorimetric assay. 190 µL of buffer containing color reagent is added to 10 µL of the sample in a transparent 96-well plate and incubated at 37°C for 30 minutes. The absorbance was measured at 600 nm using a Tecan Spark® plate reader. The amount of apolipoprotein A1 was quantified using the Apolipoprotein A1 FS assay (DiaSYS)®. It is an immunoturbidimetric test based on the interaction between the anti-apoA1 antibody and apoA1 present in the sample. 200 µL of Reagent 1 was added to 5 µL sample or control in a transparent 96-well plate. After incubating the plate at 37°C for 5 minutes, the absorbance was measured using a Tecan Spark® microplate reader at wavelength 580 nm. Next, 50 µL of Reagent 2 containing an anti-apoA1 antibody was added and the solution was incubated at 37°C for 5 minutes before measuring the absorbance at the same setting.

Apolipoprotein A1 isolation from human plasma

Human High Density Lipoprotein plasma fraction was purchased from Lee Biosolutions. Using KBr salt, the density was adjusted to 1.22–1.24 g/mL and the solution was centrifuged for 48 hours, 41,000 rpm at 4°C. The pellets were collected and diluted with Milli-Q water. Next, the HDL solution was precipitated in ice-cold methanol/chloroform (50/50 vol%). The precipitate was pelleted by centrifugation and the supernatant was decanted. The pellets were resuspended by sonication in methanol/chloroform (34/66 vol%), the solvents were decanted and the remainder was evaporated in a vacuum oven at 37°C. The resulting white/yellowish flakes were redissolved in 6M guanidine solution and buffer was exchanged for PBS via dialysis. The dialysate was collected, and concentration and quality were assessed via SDS-page, Nanodrop, Apolipoprotein FS assay, and Qtof.

In vitro gene silencing

In vitro silencing experiments were performed in a RAW264.7 cell line transfected with the pmirGLO plasmid (containing Renilla luciferase and Firefly luciferase expressing gene sequences). Cells were cultured until 80% confluency, detached with trypsin, counted, and seeded at 25,000 cells/well in a 96-well plate. After overnight recovery, the cells were transfected with nanoparticles containing either scrambled siRNA or anti Firefly luciferase siRNA at desired concentrations. After incubating for 48 hours, the medium was washed off with 1x PBS and lysate phosphate buffer (Dual-Luciferase® Reporter Assay System, Promega) was added to lyse the cells. 10 µL of the cell lysate was transferred to a white 96-well flat-bottom plate. Subsequently, 40 µL of ONE-Glo™ reagent (Dual-Luciferase® Reporter Assay System, Promega) was added and luminescence was measured with a Tecan Spark® microplate reader at an integration time of 500 ms and settle time of 1000 ms. A luminescence scan was performed to validate the intensity peak at 560 nm for Firefly luciferase. Then, 40 µL of Stop and Glo reagent (Dual-Luciferase® Reporter Assay System, Promega) was added to each well and the luminescence was measured again in the same way. The intensity peak for Renilla luciferase should be at 481 nm. The Firefly luminescence was normalized to the Renilla luminescence and subsequently expressed as a percentage of the untreated sample signal.

Cryogenic transmission electron microscopy (cryo-EM) of siRNA-aNPs

Just before the vitrification, the surface of 200-mesh lacey carbon supported copper grids (Electron Microscopy Sciences) was exposed to plasma for 40 s using a Cressington 208 carbon coater. Subsequently, 3 µL of siRNA-aNPs sample was applied on a grid and vitrified into a thin film by plunge vitrification in liquid ethane. This was done using an automated robot (FEI Vitrobot Mark IV). Cryo-EM imaging was performed on the cryoTITAN (Thermo Fisher Scientific), equipped with a field emission gun, a post column Gatan imaging filter (model 2002), and a post-GIF 2k × 2k Gatan CCD camera (model 794). The micrographs were acquired at either 6,500× (electron dose of 1.64 electrons A − 2 s − 1) or 24,000× magnification (electron dose of 11.8 A − 2 s − 1), at 300 kV acceleration voltage in bright-field TEM mode with zero-loss energy filtering and 1 s acquisition time. The size was quantified by measuring the particle diameter using ImageJ (NIH). At least 150 particles were analyzed for each formulation.

ApoA1-immunogold labeling of apoA1 on siRNA-aNPs

Maximum 0.5 h before the staining, the copper grid (carbon support film, 200 mesh, Electron Microscopy Sciences) was plasma-discharged and placed on a 20 µL droplet of the siRNA-aNP sample (dilution 1:20) for three seconds with the carbon film facing down. The grid was washed five times, by incubating on a 20 µL droplet of PBS for two minutes. Next, it was placed on 20 µL of 1% BSA in PBS for three minutes and then on 20 µL of mouse anti-human monoclonal ApoA1 antibody in 1% BSA (dilution 1:200, clone: B10, Santa Cruz Biotechnology) for 45 minutes. After washing, the grid was incubated on 20 µL of the bridging antibody in 1% BSA (1:200 dilution, polyclonal rabbit anti-mouse IgG, Jackson Immunoresearch) for 20 minutes, washed and placed on 20 µL of 10 nm protein A-coated gold nanoparticles (1:25 dilution, Cell Microscopy Core, Department of Cell Biology, Utrecht University Medical Center) for 20 minutes. Finally, the grids were washed six times in PBS and ten times in demi water (each wash was 20 µL and a three-minute incubation). Then, the grid was placed on a 20 µL droplet of 2% uranyl acetate in water for 5 minutes. After these steps, the TEM grids were air-dried overnight. The gold-labeled nanoparticles were visualized at room temperature by EM. The acquisition parameters were the same as for cryo-EM (mentioned above).

Cryo-EM analysis of lipid aggregation in siRNA-aNP formulations

The lipid aggregates were defined on cryo-EM images at 6,500 x magnification as either spherical lipid structures, larger than the main population nanoparticles and displaying different electron densities, or clusters of nanoparticles (Supplementary Fig. 6). The aggregate area was manually delineated by using a bare-hand drawing tool in ImageJ. The lipid aggregation was expressed as the percentage of aggregates in the total image area. For each formulation, at least 10 images were analyzed.

Radiolabeling of siRNA-aNPs

The used siRNA-azide had the following sequence:

5’-rArCrCrCrUrGrArArGrUrUrCrArUrCrUrGrCrArCrCrArCCG/3AzideN/-3'

5'-rCrGrGrUrGrGrUrGrGrArGrArUrGrArArCrUrUrCrArGrGrGrUrCrA-3'

siRNA-azide and DFO-DBCO were separately dissolved in MilliQ water containing 5 vol% DMSO to reach concentrations of 0.5 mg/mL. The siRNA-azide solution (270 µL, 8 nmol) was mixed with the DFO-DBCO solution (129 µL, 7.6 nmol) and incubated in the dark and at room temperature for 16 hours. The solution was dialyzed (2 kDa MWCO dialysis tube, Sigma-Aldrich) against PBS (1 L). The product was freeze-dried, and product formulation was confirmed by MALDI-TOF-MS. The siRNA-DFO remained stable for at least a year when stored at -80 °C. Next, a ⁸⁹Zr oxalate solution in 1M oxalic acid was neutralized using a 1M sodium carbonate solution until a pH between 6.8–7.4 was reached (total volume < 25 µL). The neutralized ⁸⁹Zr solution was added to DFO-siRNA dissolved in MilliQ water (0.5 mL) and the mixture was incubated at 37°C using a thermomixer (300 rpm) for 60 minutes. Completion of the reaction was confirmed by radio-TLC (Typhoon 7000IP plate reader, GE Healthcare). The ⁸⁹Zr-siRNA was directly used to formulate the ⁸⁹Zr-siRNA-aNPsm which resulted in radiochemical yields of > 80%, assessed by radio-TLC. Next, the ⁸⁹Zr-siRNA-aNPs were analyzed by CryoEM and DLS, to assess their morphology, size and dispersity, respectively.

Biodistribution

Female C57BL/6 mice were intravenously administered with ⁸⁹Zr-siRNA-aNPs (8 µCi ⁸⁹Zr correlating to 1–1.5 mg/kg siRNA) in 150–200 µL PBS via tail-vein injection. At the 24-hour time point, mice were euthanized, perfused with PBS, and tissues (bone marrow, spleen, heart, lung, muscle, tail, blood, brain, liver, and kidneys) were collected and weighed. Next, the emitted ɣ radiation was measured on a gamma counter (2480 WIZARD2 Automatic Gamma Counter, PerkinElmer). Radioactivity values were corrected for decay and normalized to tissue weight to express radioactivity concentration as a percentage injected dose per gram (%ID/g).

PET/CT imaging acquisition and analysis

PET imaging was executed 24 hours after ⁸⁹Zr-siRNA-aNPs injection using an IRIS PET/CT (Inviscan). Mice were anesthetized with a gas mixture of 2% isoflurane and 5% oxygen. A 10 minute static whole-body PET scan was conducted using an energy window between 250–750 keV followed by a 20 second whole-body CT scan (energy 80 kV, 0.9 mA, 576 projections, voxel size 160 µm). Reconstruction of the PET images was achieved with the 3D Ordered Subsets Expectation Maximization (3D-OSEM-MC) algorithm (8 subsets and 8 iterations) using decay, random, and dead-time correction. All PET and CT data were processed using OsiriX Medical Imaging software (version 13.0.3).

RNA isolation and RT-qPCR

Single cells from the bone marrow were incubated with MACS CD11b-conjugated microbeads (130-049-601, Miltenyi) for 15 minutes at 4°C. Next, magnetic bead separation was performed with LS columns and a MACS separator (Miltenyi) resulting in the collection of CD11b + cells (myeloid cells). The NucleoSpin® kit (Machery Nagel) was used to isolate RNA from 750,000 BMDMs or 5x10⁶ CD11b + cells from the bone marrow. The RNA was reverse transcribed using the iScript™ cDNA Synthesis Kit (Biorad) at 300 ng/reaction. The resulting cDNA was diluted 10x and amplified in a PowerUp™ SYBR™ Green assay (Applied Biosystems) using a QuantStudio 3 (Applied Biosystems) thermocycler. A final concentration of 100 nM for the reverse and forward primer was used; the primer details are listed in Supplementary Table 2. Thermocycling was 2 minutes at 95°C followed by 40 cycles of amplification of 15 seconds at 95°C and 30 seconds at 60°C with fluorescence acquisition at the end of each cycle. Reactions were done in duplicate and each plate included an NTC and a reverse transcriptase control. To verify the amplification of the intended product a melting curve analysis was performed from 65°C to 95°C. All amplification data was normalized to the ROX dye before further processing. Each gene of interest was normalized to two housekeeping genes and to aNP formulation containing a scrambled siRNA.

Preparing single-cell suspensions and staining for flow cytometry

Single cell suspensions were made of whole blood, spleen, and bone marrow tissue retrieved from euthanized female C57BL/6J mice. For the spleen, the entire organ was directly minced on a Corning® 70 µm cell strainer using a syringe plunger and Flow Buffer (DPBS, 0.5% BSA, 2 mM EDTA). Single-cell suspensions of the bone marrow were made via flushing the femurs with 10 mL Flow Buffer. Next, the bone marrow was immediately filtered using a 70 µm cell strainer and centrifugated at 400xg for 5 minutes at 4 ⁰C, aspirating the supernatant. The cell pellets from whole blood, spleen, and bone marrow were resuspended in 1x Red blood cell lysis (cat#420302, Biolegend) and incubated for 3 x 7 minutes on ice (blood) or 4 minutes (spleen) and 1 minute (bone marrow) at room temperature. Quenching was achieved with flow buffer and cells were centrifuged at 400xg for 5 minutes at 4°C. Next, the single cells were transferred to a Corning® V-bottom 96 well plate and incubated with 50 µL viakrome 808 (#C36628, Beckman Coulter) diluted in PBS for 20 minutes in the dark at room temperature. Next, the cells were washed, centrifuged again, and incubated with CD16/32 (Fc-block, 101302, BioLegend) antibodies (except for the progenitor staining group) for 10 minutes on ice. The cells were washed, centrifuged, and brilliant stain buffer (Invitrogen™) containing antibodies was added (total volume of 50 µL) and incubated on ice for 30 minutes in the dark. All data were acquired using a 21-color CytoFLEX LX (Beckman Coulter). The antibodies used for the myeloid cells panel and progenitor cells panel are listed below.

Myeloid panel
Target	Fluorochrome	Type	Isotype species	Isotype Ig	Isotype fragment	Clone	Immunogen	Company	Catalog number	Lot/batchnumber
CD115 (CSF-1R)	BV421	Monoclonal	Rat	IgG2a	𝛋	AFS98	-	Biolegend	135513	B334938
Ly6C	FITC	Monoclonal	Rat	IgG2c	𝛋	HK1.4	L3 cloned CTL cells	Biolegend	128006	B336286
Ly6G	APC-Cy7	Monoclonal	Rat	IgG2a	𝛋	1A8	Ly-6G transfected EL-4J cell line	Biolegend	127624	B348489
CD11b	BV785	Monoclonal	Rat	IgG2b	𝛋	M1/70	C57BL/10 splenocytes	Biolegend	101243	B373879
CD45	PerCP	Monoclonal	Rat	IgG2b	𝛋	30-F11	Mouse thymus or spleen	Biolegend	103130	B349380/ B343481
CD11c	PE-Cy7	Monoclonal	Armenian Hamster	IgG		N418	Mouse spleen dendritic cells	Biolegend	117318	B363386/B162483)
F4/80	PE	Monoclonal	Rat	IgG2a	𝛋	BM8	Murine macrophages	Biolegend	123110	B374509
LAMP1 (CD107a)	APC	Monoclonal	Rat	IgG2a	𝛋	1D4B	NIH/3T3 mouse embryonic fibroblast tissue culture cell membranes	Biolegend	121614	B338818

Progenitor panel
Target	Fluorochrome	Type	Isotype species	Isotype Ig	Isotype fragment	Clone	Immunogen	Company	Catalog number	Lot/batchnumber
CD16/32	BV510	Monoclonal	Rat	IgG2a	λ	93	Sorted pre-B cells	Biolegend	101333	B336556
CD48	APC-Cy7	Monoclonal	Armenian Hamster	IgG		HM48-1	Mouse T lymphoma MBL-2	Biolegend	103432	B329749
CD41	PE-Cy7	Monoclonal	Rat	IgG1	𝛋	MWReg30	Mouse platelets	Biolegend	133916	B293729
CD135 (Flt3/Flk2)	PerCP-eFluor 710	Monoclonal	Rat	IgG2a	𝛋	A2F10		ThermoFisher	46-1351-82	2202073
CD150	BV605	Monoclonal	Rat	IgG2a	λ	TC15-12F12.2	Mouse SLAM-human IgG1 fusion protein	Biolegend	115927	B361886
LAMP1 (CD107a)	APC	Monoclonal	Rat	IgG2a	𝛋	1D4B	NIH/3T3 mouse embryonic fibroblast tissue culture cell membranes	Biolegend	121614	B338818
Lineage cocktail: CD3 + CD11b + CD45R/B220 + Ly-76 + Ly6G + Ly6C	FITC							Biolegend
CD34	BV421	Monoclonal	Rat	IgG2a	𝛋	SA376A4	Mouse CD34 transfected cells.	Biolegend	152208	B378790
CD117 (c-kit)	BV785	Monoclonal	Rat	IgG2b	𝛋	ACK2		Biolegend	135138	B387239
Ly-6A/E (Sca-1)	PE	Monoclonal	Rat	IgG2a	𝛋	W18174A	EL4 cell	Biolegend	160906	B370192

Clinical chemistry and cytokine measurements

Female C57BL/6J mice were intravenously administrated with 0.5 mg/kg siRNA or PBS (n = 4 per group). After 4 hours, serum was collected and cytokines (IL-6 and TNF-α) levels were measured using enzyme-linked immunosorbent assay (ELISA) kits (ELISA MAX™ Deluxe Set Mouse IL-6 (Biolegend) and ELISA MAX™ Deluxe Set Mouse TNF-α, Biolegend) according to the manufacturer’s instructions. After 24 hours and 72 hours, whole blood was collected in BD Vacutainer® Heparin Tubes (Becton Dickinson) and serum was separated. The liver function (ASAT and ALAT) and renal function (urea and creatinine) were measured at the Radboudumc Laboratorium voor Diagnostiek core facility.

Inbred mice strain

All animal experiments were executed complying with both European and Dutch guidelines according to the care and use of laboratory mice after experimental approval by Radboud University Medical Center’s Dierexperimentencommissie (DEC) (CCD: AVD10300 2021 15550). 8–12 weeks old female C57BL/6J mice (Mus musculus) obtained from Charles River Germany were co-housed in climate-controlled conditions (temperature of 20–24°C and a humidity level of 45–65% RV). An acclimatization period of 1 week was conducted and all mice had bare access to food and water. Mice were randomized and assigned to control and treatment groups.

Tumor inoculation and treatment regimen

Female C57BL/6J mice were inoculated with 1x10⁵ B16F10 melanoma cells in 100 µL PBS supplemented with 0.5% fetal bovine serum (FBS) via subcutaneous injection in the right flank on day − 7. Treatment for all experiments consisted of intravenous administration (6 x 0.5 mg/kg in 9 days, 36 hours apart) of siRNA_scrambled-aNP₁₈ or siRNA_SHP2-aNP₁₈. Treatment started on day 7 and lasted until day 17.

Statistical analysis

All data are presented as mean ± standard deviation (SD) and analyzed using GraphPad Prism 10.0 by one-way ANOVA or student t-test, as indicated with all details in the figure legends. p values < 0.05 were considered significant, with levels of significance indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Acknowledgements

This work was supported by a Dutch Research Council (NWO) Vidi grant (no. 19681) to R.v.d.M, and an NWO Vici grant (no. 91818622) and ERC Advanced grant (no. 101019807) to W.J.M.M.

Competing interests

M.G.N. and W.J.M.M. are scientific co-founders of and have equity in Trained Therapeutix Discovery. W.J.M.M. is CSO of Trained Therapeutix Discovery. M.G.N. and W.J.M.M. are scientific co-founders of and have equity in BioTrip. S.R.J.H., T.A., R.Z., H.M.J., P.M.F., W.J.M.M., E.K., and R.v.d.M are listed as inventors on patent application WO2022268913A1 related to this manuscript.

Kulkarni, J. A. et al. The current landscape of nucleic acid therapeutics. Nat Nanotechnol 16, 630–643 (2021).
Akinc, A. et al. The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs. Nat Nanotechnol 14, 1084–1087 (2019).
Adams, D. et al. Patisiran, an RNAi Therapeutic, for Hereditary Transthyretin Amyloidosis. New England Journal of Medicine 379, 11–21 (2018).
Polack, F. P. et al. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. New England Journal of Medicine 383, 2603–2615 (2020).
Baden, L. R. et al. Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. New England Journal of Medicine 384, 403–416 (2021).
Witzigmann, D. et al. Lipid nanoparticle technology for therapeutic gene regulation in the liver. Adv Drug Deliv Rev 159, 344–363 (2020).
Cheng, Q. et al. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR–Cas gene editing. Nat Nanotechnol 15, 313–320 (2020).
Dilliard, S. A., Cheng, Q. & Siegwart, D. J. On the mechanism of tissue-specific mRNA delivery by selective organ targeting nanoparticles. Proceedings of the National Academy of Sciences 118, e2109256118 (2021).
Kedmi, R. et al. A modular platform for targeted RNAi therapeutics. Nat Nanotechnol 13, 214–219 (2018).
Veiga, N. et al. Cell specific delivery of modified mRNA expressing therapeutic proteins to leukocytes. Nat Commun 9, 4493 (2018).
Tarab-Ravski, D. et al. Delivery of Therapeutic RNA to the Bone Marrow in Multiple Myeloma Using CD38‐Targeted Lipid Nanoparticles. Advanced Science 10, 2301377 (2023).
Tombácz, I. et al. Highly efficient CD4 + T cell targeting and genetic recombination using engineered CD4 + cell-homing mRNA-LNPs. Molecular Therapy 29, 3293–3304 (2021).
Parhiz, H. et al. PECAM-1 directed re-targeting of exogenous mRNA providing two orders of magnitude enhancement of vascular delivery and expression in lungs independent of apolipoprotein E-mediated uptake. J Control Release 291, 106–115 (2018).
Rurik, J. G. et al. CAR T cells produced in vivo to treat cardiac injury. Science 375, 91–96 (2022).
Lokugamage, M. P., Sago, C. D., Gan, Z., Krupczak, B. R. & Dahlman, J. E. Constrained Nanoparticles Deliver siRNA and sgRNA to T Cells In Vivo without Targeting Ligands. Adv Mater 31, e1902251 (2019).
Da Silva Sanchez, A. J. et al. Universal Barcoding Predicts In Vivo ApoE-Independent Lipid Nanoparticle Delivery. Nano Lett 22, 4822–4830 (2022).
Wasan, K. M., Brocks, D. R., Lee, S. D., Sachs-Barrable, K. & Thornton, S. J. Impact of lipoproteins on the biological activity and disposition of hydrophobic drugs: implications for drug discovery. Nat Rev Drug Discov 7, 84–99 (2008).
Vickers, K. C., Palmisano, B. T., Shoucri, B. M., Shamburek, R. D. & Remaley, A. T. MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol 13, 423–433 (2011).
Huang, R. et al. Apolipoprotein A-I structural organization in high-density lipoproteins isolated from human plasma. Nat Struct Mol Biol 18, 416–423 (2011).
Mulder, W. J. M. et al. High-Density Lipoprotein Nanobiologics for Precision Medicine. Acc Chem Res 51, 127–137 (2018).
Schrijver, D. P. et al. Nanoengineering Apolipoprotein A1-Based Immunotherapeutics. Adv Ther (Weinh) 4, 2100083 (2021).
Mulder, W. J. M., Ochando, J., Joosten, L. A. B., Fayad, Z. A. & Netea, M. G. Therapeutic targeting of trained immunity. Nat Rev Drug Discov 18, 553–566 (2019).
van Leent, M. M. T. et al. Regulating trained immunity with nanomedicine. Nat Rev Mater 7, 465–481 (2022).
Schrijver, D. P. et al. Resolving sepsis-induced immunoparalysis via trained immunity by targeting interleukin-4 to myeloid cells. Nat Biomed Eng 1–16 (2023) doi:10.1038/s41551-023-01050-0.
Jonas, A., Zorich, N. L., Kézdy, K. E. & Trick, W. E. Reaction of discoidal complexes of apolipoprotein A-I and various phosphatidylcholines with lecithin cholesterol acyltransferase. Interfacial effects. J Biol Chem 262, 3969–74 (1987).
Paunovska, K. et al. A Direct Comparison of in Vitro and in Vivo Nucleic Acid Delivery Mediated by Hundreds of Nanoparticles Reveals a Weak Correlation. Nano Lett 18, 2148–2157 (2018).
Chen, H. et al. SHP2 is a multifunctional therapeutic target in drug resistant metastatic breast cancer. Oncogene 39, 7166–7180 (2020).
Christofides, A. et al. SHP-2 and PD-1-SHP-2 signaling regulate myeloid cell differentiation and antitumor responses. Nat Immunol 24, 55–68 (2023).
Yvan-Charvet, L. et al. ATP-binding cassette transporters and HDL suppress hematopoietic stem cell proliferation. Science (1979) 328, 1689–1693 (2010).
Silvestre-Roig, C., Braster, Q., Ortega-Gomez, A. & Soehnlein, O. Neutrophils as regulators of cardiovascular inflammation. Nat Rev Cardiol 17, 327–340 (2020).
Bhargava, S., de la Puente-Secades, S., Schurgers, L. & Jankowski, J. Lipids and lipoproteins in cardiovascular diseases: a classification. Trends Endocrinol Metab 33, 409–423 (2022).
Pownall, H. J., Rosales, C., Gillard, B. K. & Gotto, A. M. High-density lipoproteins, reverse cholesterol transport and atherogenesis. Nat Rev Cardiol 18, 712–723 (2021).
Yang, M. et al. Efficient Cytosolic Delivery of siRNA Using HDL-Mimicking Nanoparticles. Small 7, 568–573 (2011).
Wolfrum, C. et al. Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Nat Biotechnol 25, 1149–1157 (2007).
Shahzad, M. M. K. et al. Targeted Delivery of Small Interfering RNA Using Reconstituted High-Density Lipoprotein Nanoparticles. Neoplasia 13, 309-IN8 (2011).
Fischer, N. O. et al. Colocalized delivery of adjuvant and antigen using nanolipoprotein particles enhances the immune response to recombinant antigens. J Am Chem Soc 135, 2044–2047 (2013).
Kuai, R., Ochyl, L. J., Bahjat, K. S., Schwendeman, A. & Moon, J. J. Designer vaccine nanodiscs for personalized cancer immunotherapy. Nat Mater 16, 489–496 (2017).
Krohn-Grimberghe, M. et al. Nanoparticle-encapsulated siRNAs for gene silencing in the haematopoietic stem-cell niche. Nat Biomed Eng 4, 1076–1089 (2020).
Sago, C. D. et al. Nanoparticles that deliver RNA to bone marrow identified by in vivo directed evolution. J Am Chem Soc 140, 17095–17105 (2018).
Guimarães, P. P. G. et al. In vivo bone marrow microenvironment siRNA delivery using lipid–polymer nanoparticles for multiple myeloma therapy. Proceedings of the National Academy of Sciences 120, e2215711120 (2023).
Shi, D., Toyonaga, S. & Anderson, D. G. In Vivo RNA Delivery to Hematopoietic Stem and Progenitor Cells via Targeted Lipid Nanoparticles. Nano Lett 23, 2938–2944 (2023).
Breda, L. et al. In vivo hematopoietic stem cell modification by mRNA delivery. Science (1979) 381, 436–443 (2023).

Yes there is potential Competing Interest. M.G.N. and W.J.M.M. are scientific co-founders of and have equity in Trained Therapeutix Discovery. W.J.M.M. is CSO of Trained Therapeutix Discovery. M.G.N. and W.J.M.M. are scientific co-founders of and have equity in BioTrip. S.R.J.H., T.A., R.Z., H.M.J., P.M.F., W.J.M.M., E.K., and R.v.d.M are listed as inventors on patent application WO2022268913A1 related to this manuscript.

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A nature-inspired nanodelivery platform for gene silencing in hematopoietic stem and progenitor cells

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Abstract

Figures

Introduction

Prototyping apolipoprotein lipid nanoparticles for siRNA immune cell delivery

Establishing and characterizing an apolipoprotein lipid nanoparticle library

In-depth profiling of lead siRNA apolipoprotein lipid nanoparticle

Discussion

Materials and methods

Materials

siRNA-aNP production

siRNA-aNP physicochemical analysis

Apolipoprotein A1 isolation from human plasma

Cryogenic transmission electron microscopy (cryo-EM) of siRNA-aNPs

ApoA1-immunogold labeling of apoA1 on siRNA-aNPs

Cryo-EM analysis of lipid aggregation in siRNA-aNP formulations

Radiolabeling of siRNA-aNPs

Biodistribution

PET/CT imaging acquisition and analysis

RNA isolation and RT-qPCR

Preparing single-cell suspensions and staining for flow cytometry

Clinical chemistry and cytokine measurements

Inbred mice strain

Tumor inoculation and treatment regimen

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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