Oligonucleotide therapeutics have shown promise as a diverse treatment option for a wide range of diseases. Developing successful oligonucleotide therapeutics depends on being able to efficiently deliver the therapeutic to the target cells.
There are various delivery methods that can be used, one being adeno-associated viruses (see our blog post here). Another, and more common, delivery system is the use of lipid nanoparticles (LNPs).
Today, the typical LNP composition consists of phospholipids, cholesterol, ionizable lipids and PEG-lipids to strengthen stability and aid in endosomal release[1]. The large majority of LNPs biodistribution is primarily to the liver, partly due to the binding of proteins, such as apolipoprotein (ApoE), allowing for hepatic uptake via cellular receptors (low-density lipoprotein receptor; LDLR). Given this, significant research into LNP formulations focuses on modifications to either improve the delivery and release in hepatocytes or to target alternate organ/cell types. However, with modifications there is concern of toxic accumulation in the liver. Therefore, pre-clinical screening is key to predict novel LNPs clinical success.
Pre-clinical animal models are important for therapeutic development, with the goal of accurately predicting the efficacy and toxicity of novel therapeutics prior to transitioning to the clinic. In the case of LNP development, non-human primate (NHP) models typically can predict LNP delivery and safety, however screening LNPs in this model is not feasible due to the high cost and ethical concerns. Rodents are more accessible and cost effective however, murine hepatocytes do not accurately predict delivery and efficiency of human-targeted LNP therapeutics. Whereas, the PXB-mouse humanized liver model offers the advantage of predicting not only the delivery specifically to human hepatocytes but also the translational efficiency of the oligonucleotide with the convenience of a small animal model.
PXB-mice can be used to accurately assess human hepatotoxicity, delivery efficiency, and translational efficacy of LNP therapeutics.
PXB-mouse livers are engrafted with human hepatocytes and have stable expression of human genes and proteins, making them an ideal model to test the efficacy of human-specific targets. Furthermore, PXB-mice have functional human transporters and enzymes as well as human-like lipoprotein profiles. Here we highlight research that focuses on LNP delivery and efficiency in the PXB-mouse model.
To better understand the potential of PXB-mice as a platform for investigating LNP therapeutics, Acuitas Therapeutics explored the delivery, distribution, efficiency, and tolerability of three of their LNP formulations (LNP07, LNP09, and LNP13)[2]. In order to assess delivery, each LNP encapsulated a reporter IgG mRNA. Delivery efficiency to human hepatocytes was LNP specific, with LNP07 and LNP13 outperforming LNP09 (Figure 1). However, there was also delivery to the remaining mouse hepatocytes with no clear human specific uptake for any of the LNPs tested.
Figure 1: Immunohistochemistry (IHC) and hematoxylin-eosin (H&E) staining of liver sections from PXB-mice dosed with LNP09, LNP07, or LNP13. Samples were collected 24-hours post-LNP administration. IgG protein expression (LNP payload) was observed in regions of human hepatocytes (identified by the expression of a human-specific hepatocyte marker) for LNP07 and LNP13, but was not as robust for LNP09.
Figure 2:PXB-mice were intravenously dosed with LNP07, LNP09, and LNP13 at different dose levels. Serum IgG protein levels were assessed at 24-hours post-dose.
To determine the LNPs potency, serum IgG levels were assessed and based on expression, LNPs were ranked. LNP13 outperformed the other LNPs, but was closely followed by LNP07 whereas LNP09 had minimal IgG levels detected at the 3 mg/kg dose level (Figure 2). Blood biochemistry analysis showed dose-dependent increases in total alanine transaminase (ALT), human ALT (hALT), and aspartate aminotransferase (AST). Total bilirubin (TBIL) levels were not significantly altered across all dose levels (Figure 3). While there were increases in liver values (ALT and AST), the LNPs were well tolerated in PXB-mice with no observed clinical manifestations even at the highest dose (5 mg/kg for LNP13).
Moving forward with the top performing formulation, LNP13, murine- and human-specific delivery was assessed using quantitative mass spectrometry imaging (QMSI). LNP-derived aminolipid levels were observed throughout the PXB-mouse liver with no significant differences between human and mouse hepatocyte uptake (Figure 4).
Figure 4: Quantitative mass spectrometry imaging was performed on liver tissue samples from PXB-mice dosed with different dose levels of LNP13 at 24-hours post-administration. Mouse and human regions were identified by staining for a human-specific hepatocyte marker. Aminolipid values in the entire liver section are bolded, and mouse hepatocyte values are in gray, and human hepatocyte values are in orange.
Figure 5: Plasma aminolipid levels were measured at different time points following LNP13 (1.5 mg/kg) intravenous administration in PXB-mice.
Looking at the pharmacokinetics of LNP13, there is rapid clearance of plasma LNP-derived aminolipid levels within hours of dosing and the clearance rate slows over the observation period (Figure 5). Importantly, there is extensive distribution of IgG mRNA throughout the liver demonstrating effective hepatic delivery of LNP13 in the PXB-mouse (Figure 6). IgG protein levels were observed in mouse regions as early as 3 hours post-dose but was slightly delayed in human hepatocytes. However, by 24 to 48 hours post-dose the apparent difference in expression between mouse and human hepatocytes was diminished (Figure 7).
Taken together, these LNP formulations were well tolerated in PXB-mice. Furthermore, there was robust delivery to the liver with no preference between human and mouse hepatocytes although the efficiency is LNP-dependent. While IgG mRNA was broadly distributed, delayed protein expression in human hepatocytes suggests a potential difference in LNP uptake, endosomal escape, and/or translation compared to mouse hepatocytes.
Figure 8: Biodistribution of siRNA-LNP detected by ex vivo Cy3 imaging of PXB-mouse liver, spleen, kidney, lung, and heart 4 hours post-administration.
Recently, the overall biodistribution of LNP in the PXB-mouse model was shown to target predominately the liver[3]. PXB-mice injected with LNP containing Cy3-siLuc had high levels of Cy3 luminescence observed in the liver (Figure 8). In fact, over 90% of fluorescence was detected in the liver with minimal delivery to other organs such as the spleen (<5%), demonstrating liver-specific delivery for this LNP.
Overall, these studies emphasize the value of the humanized liver mouse model in generating translationally relevant results, which can guide the selection of effective siRNA delivery methods.
As mentioned previously, while NHP models outperform wild type rodent models for predicting LNP delivery and efficiency they come at a high cost, both financially and ethically. Furthermore, although NHP are genetically similar to humans, slight genetic differences may reduce the ability to predict efficacy for certain human-specific genes. Therefore, having a more cost-effective model that predicts efficiency and safety for human-specific LNP therapeutics is important.
We have previously highlighted the PXB-mouse as an alternative model to NHPs (see our blog post here). In regards to LNP delivery, similar protein suppression was observed in PXB-mice and NHPs following LNP delivery of a microRNA (miR) target site (miRts) for miR122, demonstrating that PXB-mice can be used for pre-clinical screening of LNP therapeutics (Figure 9)[4].
To investigate the effectiveness of the siRNA-LNP delivery method, PXB-mice were injected with siRNA-LNP to knockdown the expression of transthyretin (Ttrl/TTR)[3]. Transthyretin is primarily expressed by hepatocytes and is secreted into the bloodstream allowing for in vivo monitoring of knockdown efficiency. There was a significant reduction in plasma human TTR levels observed at 1-, 3-, and 7-days post-injection with siRNA targeting human TTR (hTTR)-LNP (Figure 10)[3]. Additionally, hepatic hTTR mRNA levels were significantly reduced in a dose-dependent manner. Whereas, mouse TTR protein levels were not affected, demonstrating the ability to specifically target human genes in the PXB-mouse.
Figure 11: PXB-mice treated with hetero-gapmer had significant reductions in body weight and blood human albumin (h-Alb) levels.
Importantly, the PXB-mouse model can predict human-specific hepatoxicity. In a hepatitis B virus (HBV) study, hetero-gapmer targeting a host factor (dedicator of cytokinesis 11; DOCK11) was used in the AAV-HBV mouse model. This treatment was well tolerated and effective in suppressing HBV in this model. However, when tested in the PXB-mouse there were no changes observed for HBV parameters and there were signs of liver toxicity observed in the gapmer control animals (Figure 11)[5]. Due to the observed human-specific hepatotoxicity, an LNP encapsulated siRNA targeting DOCK11 was generated and tested. In HBV-infected PXB-mice treated with LNP-siRNA DOCK11 there was a significant reduction in HBV DNA and cccDNA levels (Figure 12)[5]. Unlike gapmer treatment, the LNP-siRNA had no effect on body weight or human albumin levels demonstrating that this therapeutic is well tolerated (Figure 12)[5].
These data provide evidence that PXB-mice can be used to predict LNP delivery and efficiency. Due to the high-degree of humanization of the liver, targeting human-specific genes is made possible in this convenient animal model, allowing for researchers to predict promising clinical LNP candidates. Contact our scientific team today to harness the translational capability of the PXB-mouse.