Abstract PR04: CRISPR lipid nanoparticle modulates the tumor immune microenvironment of liver metastases by genetically eliminating M2 polarization pathway in macrophages
Authors: Galbraith Tyler , Cook Harlan , Ziemys Arturas , Leonard Fransisca 
Published: September 2022

Liver metastasis carries a poor prognosis, specifically due to impaired transport of chemotherapies into the lesions causing insufficient drug accumulation which leads to poor treatment response. The tumor immune microenvironment (TIME), especially macrophages which constitute about 10% of total population in the liver, has been recognized as a major factor in supporting tumor growth and affecting therapeutic outcomes. Repolarization of pro- tumorigenic macrophages to anti-tumorigenic M1 macrophages has proven to be a promising new immunotherapy approach. We have created a lipid nanoparticle (LNP) platform capable of delivering a Cas12a/RICTOR-crRNA complex to macrophages to genetically block their differentiation to an M2 phenotype. The particle consists of a mix of phospholipid materials that encapsulates the CRISPR complex and is generated through hydration/extrusion. The resulting biocompatible nanocarriers are around 130 nm, -9 mV zeta potential, and have an encapsulation efficiency of &gt;80%. We administered the CRISPR-LNP via intravenous injection on a syngeneic liver metastasis mouse model. Single- and multi-dose studies were conducted to determine biodistribution, efficacy, and survivability. Lastly, a digital pathology analysis of immunohistochemical images allowed for investigation of the in situ interactions amongst immune and tumor cells within the TIME. Biodistribution data of our rhodamine-lipid-tagged LNP from IVIS imaging and organ homogenate analysis showed that between 1- and 24-hours post-injection, the LNP was predominantly distributed to the liver. The amount of LNP peaked in the liver at around 1 hour post-injection, and by 4 hours the LNP had largely cleared the body. However, signal from the homogenization of isolated tumor metastases remained steady over time despite the decline in the liver signal. Furthermore, intravital microscopy analysis showed rhodamine signal isolated within tumor sites after 24 hours despite the lack of proper vascularization, suggesting the macrophage’s ability to take up particles and infiltrate hypovascularized tumor regions. IHC analysis of liver sections using CD80, CD206, and F4/80 markers showed an increase in M1:M2 ratio following LNP administration when compared to control. Digital pathology analysis on IHC and Imaging Mass Cytometry data showed a decrease in Ki67 expression in 4T1 cells as well as an increased number of immune cells, in particular macrophages, CD8+ and CD8+/PD1+ T-cells. Our results show that reprograming macrophages is a powerful tool to suppress cancer cell growth and increase the presence of cytotoxic CD8+ T- cells. Thus, we hypothesize that this therapy can potentially be administered alongside current leading immunotherapies to obtain synergistic effects. We are currently conducting multi-dose efficacy and survival studies, and aim to combine the CRISPR-LNP treatment with anti PD-1 antibodies to observe their efficacy to protect against resistance. Ultimately, this novel CRISPR vehicle has applications to other cancers with similar molecular/inflammation signature.

Citation Format: Tyler Galbraith, Harlan Cook, Arturas Ziemys, Fransisca Leonard. CRISPR lipid nanoparticle modulates the tumor immune microenvironment of liver metastases by genetically eliminating M2 polarization pathway in macrophages [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PR04.