Chimeric mouse model for MRI contrast agent evaluation.

PURPOSE: While rodents are the primary animal models for contrast agent evaluation, rodents can potentially misrepresent human organ clearance of newly developed contrast agents. For example, gadolinium (Gd)-BOPTA has ~50% hepatic clearance in rodents, but ~5% in humans. This study demonstrates the benefit of chimeric mice expressing human hepatic OATPs (organic anion-transporting polypeptides) to improve evaluation of novel contrast agents for clinical use. METHODS: FVB (wild-type) and OATP1B1/1B3 knock-in mice were injected with hepatospecific MRI contrast agents (Gd-EOB-DTPA, Gd-BOPTA) and nonspecific Gd-DTPA. T1 -weighted dynamic contrast-enhanced MRI was performed on mice injected intravenously. Hepatic MRI signal enhancement was calculated per time point. Mass of gadolinium cleared per time point and percentage elimination by means of feces and urine were also measured. RESULTS: Following intravenous injection of Gd-BOPTA in chimeric OATP1B1/1B3 knock-in mice, hepatic MRI signal enhancement and elimination by liver was more reflective of human hepatic clearance than that measured in wild-type mice. Gd-BOPTA hepatic MRI signal enhancement was reduced to 22% relative to wild-type mice. Gd-BOPTA elimination in wild-type mice was 83% fecal compared with 32% fecal in chimeric mice. Hepatic MRI signal enhancement and elimination for Gd-EOB-DTPA and Gd-DTPA were similar between wild-type and chimeric cohorts. CONCLUSION: Hepatic MRI signal enhancement and elimination of Gd-EOB-DTPA, Gd-BOPTA, and Gd-DTPA in chimeric OATP1B1/1B3 knock-in mice closely mimics that seen in humans. This study provides evidence that the chimeric knock-in mouse is a more useful screening tool for novel MRI contrast agents destined for clinical use as compared to the traditionally used wild-type models.

Inhaled Nanoformulated mRNA Polyplexes for Protein Production in Lung Epithelium.

Noninvasive aerosol inhalation is an established method of drug delivery to the lung, and remains a desirable route for nucleic-acid-based therapeutics. In vitro transcribed (IVT) mRNA has broad therapeutic applicability as it permits temporal and dose-dependent control of encoded protein expression. Inhaled delivery of IVT-mRNA has not yet been demonstrated and requires development of safe and effective materials. To meet this need, hyperbranched poly(beta amino esters) (hPBAEs) are synthesized to enable nanoformulation of stable and concentrated polyplexes suitable for inhalation. This strategy achieves uniform distribution of luciferase mRNA throughout all five lobes of the lung and produces 101.2 ng g-1 of luciferase protein 24 h after inhalation of hPBAE polyplexes. Importantly, delivery is localized to the lung, and no luminescence is observed in other tissues. Furthermore, using an Ai14 reporter mouse model it is identified that 24.6% of the total lung epithelial cell population is transfected after a single dose. Repeat dosing of inhaled hPBAE-mRNA generates consistent protein production in the lung, without local or systemic toxicity. The results indicate that nebulized delivery of IVT-mRNA facilitated by hPBAE vectors may provide a clinically relevant delivery system to lung epithelium.

Synthesis and Biological Evaluation of Ionizable Lipid Materials for the In Vivo Delivery of Messenger RNA to B Lymphocytes.

B lymphocytes regulate several aspects of immunity including antibody production, cytokine secretion, and T-cell activation; moreover, B cell misregulation is implicated in autoimmune disorders and cancers such as multiple sclerosis and non-Hodgkin's lymphomas. The delivery of messenger RNA (mRNA) into B cells can be used to modulate and study these biological functions by means of inducing functional protein expression in a dose-dependent and time-controlled manner. However, current in vivo mRNA delivery systems fail to transfect B lymphocytes and instead primarily target hepatocytes and dendritic cells. Here, the design, synthesis, and biological evaluation of a lipid nanoparticle (LNP) system that can encapsulate mRNA, navigate to the spleen, transfect B lymphocytes, and induce more than 60 pg of protein expression per million B cells within the spleen is described. Importantly, this LNP induces more than 85% of total protein production in the spleen, despite LNPs being observed transiently in the liver and other organs. These results demonstrate that LNP composition alone can be used to modulate the site of protein induction in vivo, highlighting the critical importance of designing and synthesizing new nanomaterials for nucleic acid delivery.

Identification of Novel Fibrosis Modifiers by In Vivo siRNA Silencing.

Fibrotic diseases contribute to 45% of deaths in the industrialized world, and therefore a better understanding of the pathophysiological mechanisms underlying tissue fibrosis is sorely needed. We aimed to identify novel modifiers of tissue fibrosis expressed by myofibroblasts and their progenitors in their disease microenvironment through RNA silencing in vivo. We leveraged novel biology, targeting genes upregulated during liver and kidney fibrosis in this cell lineage, and employed small interfering RNA (siRNA)-formulated lipid nanoparticles technology to silence these genes in carbon-tetrachloride-induced liver fibrosis in mice. We identified five genes, Egr2, Atp1a2, Fkbp10, Fstl1, and Has2, which modified fibrogenesis based on their silencing, resulting in reduced Col1a1 mRNA levels and collagen accumulation in the liver. These genes fell into different groups based on the effects of their silencing on a transcriptional mini-array and histological outcomes. Silencing of Egr2 had the broadest effects in vivo and also reduced fibrogenic gene expression in a human fibroblast cell line. Prior to our study, Egr2, Atp1a2, and Fkbp10 had not been functionally validated in fibrosis in vivo. Thus, our results provide a major advance over the existing knowledge of fibrogenic pathways. Our study is the first example of a targeted siRNA assay to identify novel fibrosis modifiers in vivo.

Barcoded nanoparticles for high throughput in vivo discovery of targeted therapeutics.

Nucleic acid therapeutics are limited by inefficient delivery to target tissues and cells and by an incomplete understanding of how nanoparticle structure affects biodistribution to off-target organs. Although thousands of nanoparticle formulations have been designed to deliver nucleic acids, most nanoparticles have been tested in cell culture contexts that do not recapitulate systemic in vivo delivery. To increase the number of nanoparticles that could be tested in vivo, we developed a method to simultaneously measure the biodistribution of many chemically distinct nanoparticles. We formulated nanoparticles to carry specific nucleic acid barcodes, administered the pool of particles, and quantified particle biodistribution by deep sequencing the barcodes. This method distinguished previously characterized lung- and liver- targeting nanoparticles and accurately reported relative quantities of nucleic acid delivered to tissues. Barcode sequences did not affect delivery, and no evidence of particle mixing was observed for tested particles. By measuring the biodistribution of 30 nanoparticles to eight tissues simultaneously, we identified chemical properties promoting delivery to some tissues relative to others. Finally, particles that distributed to the liver also silenced gene expression in hepatocytes when formulated with siRNA. This system can facilitate discovery of nanoparticles targeting specific tissues and cells and accelerate the study of relationships between chemical structure and delivery in vivo.

Efficacy and immunogenicity of unmodified and pseudouridine-modified mRNA delivered systemically with lipid nanoparticles in vivo.

mRNA has broad potential for treating diseases requiring protein expression. However, mRNA can also induce an immune response with associated toxicity. Replacement of uridine bases with pseudouridine has been postulated to modulate both mRNA immunogenicity and potency. Here, we explore the immune response and activity of lipid nanoparticle-formulated unmodified and pseudouridine-modified mRNAs administered systemically in vivo. Pseudouridine modification to mRNA had no significant effect on lipid nanoparticle physical properties, protein expression in vivo, or mRNA immunogenicity compared to unmodified mRNA when delivered systemically with liver-targeting lipid nanoparticles, but reduced in vitro transfection levels. Indicators of a transient, extracellular innate immune response to mRNA were observed, including neutrophilia, myeloid cell activation, and up-regulation of four serum cytokines. This study provides insight into the immune responses to mRNA lipid nanoparticles, and suggests that pseudouridine modifications may be unnecessary for therapeutic application of mRNA in the liver.

Optimization of Lipid Nanoparticle Formulations for mRNA Delivery in Vivo with Fractional Factorial and Definitive Screening Designs.

Intracellular delivery of messenger RNA (mRNA) has the potential to induce protein production for many therapeutic applications. Although lipid nanoparticles have shown considerable promise for the delivery of small interfering RNAs (siRNA), their utility as agents for mRNA delivery has only recently been investigated. The most common siRNA formulations contain four components: an amine-containing lipid or lipid-like material, phospholipid, cholesterol, and lipid-anchored polyethylene glycol, the relative ratios of which can have profound effects on the formulation potency. Here, we develop a generalized strategy to optimize lipid nanoparticle formulations for mRNA delivery to the liver in vivo using Design of Experiment (DOE) methodologies including Definitive Screening and Fractional Factorial Designs. By simultaneously varying lipid ratios and structures, we developed an optimized formulation which increased the potency of erythropoietin-mRNA-loaded C12-200 lipid nanoparticles 7-fold relative to formulations previously used for siRNA delivery. Key features of this optimized formulation were the incorporation of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and increased ionizable lipid:mRNA weight ratios. Interestingly, the optimized lipid nanoparticle formulation did not improve siRNA delivery, indicating differences in optimized formulation parameter design spaces for siRNA and mRNA. We believe the general method described here can accelerate in vivo screening and optimization of nanoparticle formulations with large multidimensional design spaces.