Swine reporter model for preclinical evaluation and characterization of gene delivery vectors

Abstract

Delivery of gene therapy vectors targeted to any somatic cell remains a key barrier for the development of genetic medicines. While rodent models provide insights into vector biodistribution and cellular tropism, their anatomical and physiological differences from humans limit their translational potential and studies in large animal models are often required. In this study, we developed a swine reporter model (SRM-1) to evaluate both viral and nonviral vector delivery in a large animal system. The SRM-1 model harbors a Cre- and CRISPR-activated tdTomato reporter at the Rosa26 locus that allows for tracing of cell-specific delivery and expression of gene therapy vectors in vivo. To evaluate this model, we administered adeno-associated virus serotype 9 (AAV9) and lipid nanoparticles (LNPs) carrying mRNA systemically and found successful in vivo reporter activation across a variety of tissues. Intracerebroventricular (ICV) administration of LNP-Cre mRNA was also performed and demonstrated localized activation in cortical brain cells. In addition to biodistribution studies, this model has utility for testing safety and clinically relevant administration methods, surgical and non-surgical, of delivery vectors. Our findings support the SRM-1 model as a valuable tool for advancing gene therapies from preclinical testing to clinical application.

Fig. 1. Generation of the SRM-1 Cre- or CRISPR-inducible swine reporter model.

(A) Schematic illustration of the SRM-1 reporter integrated into the swine Rosa26 gene. Reporter design includes loxP sites (purple triangles) and CRISPR sites (blue and red arrows) for Cre- or genome editing-mediated reporter activation, respectively. (B) PCR of the integration junction of the SRM-1 construct in the Rosa26 gene and SRM-1 construct copy number measured by ddPCR in founder SRM-1 animals. Asterisk indicates an animal with imprecise reporter integration. (C) %tdTomato activation in SRM-1 fibroblasts measured by flow cytometry after transfection with either Cre mRNA, SpCas9 RNP, or AsCasl2a RNP. Closed circles are individual data points, error bars are standard error of the mean.

Fig. 2. SRM-1 detects recombination in vivo by intravascular delivery of AAV9-Cre.

(A) Schematic illustration of systemic AAV9 in vivo study. (B) %SRM-1 recombination rates of the loxP sites in tissues measured by ddPCR. Open circles (purple bars) are an AAV-injected animal (n=1), closed circles are an uninjected animal (n=1). (C) Immunohistochemistry of tissues taken from an injected and uninjected SRM-1 piglet. Tissues were co-stained for tdTomato and sodium iodide symporter (NIS) and detected with DAB. Scale bar, 300 μm.

Fig. 3. Serum chemistry and blood counts of LNP-injected piglets.

(A) LNP-mRNA delivery study design schematic. (B) LNP formulation schematic of mRNA using microfluidic mixing. (C) Absolute values of serum ALT, AST, creatinine, creatinine kinase, and albumin as well as blood cell counts (white blood cells, lymphocytes, neutrophils, red blood cells) in three LNP-injected SRM-1 pigs (purple), one mock-injected SRM-1 pig (grey), and 2 non-injected wild type sibling piglets (black). The red dashed lines are the high and low references ranges supplied by the diagnostic laboratory. One blood sample per piglet was taken at each time point.

Fig. 4. Tissue and cellular transfection by systemic LNP-mRNA can be tracked using SRM-1 swine after multiple weeks.

(A) Recombination of SRM-1 genomic DNA collected from LNP-injected SRM-1 pigs (n=3, 1 male and 2 female) and a mock-injected male SRM-1 pig (n=1). Open circles are individual animal measurements for LNP-injected animals, closed circles are for mock-injected. Error bars are standard error of the mean. (B) Distribution of LNP in each swine liver lobe. R = right lateral lobe, RM = right medial lob, LM = left medial lob, L = left lateral lobe. Open circles are individual animal measurements. (C) Fluorescent histological images of SRM-1 tissues with native tdTomato-producing cells (red) and stained nuclei (blue) after LNP-mRNA (Cre) injections. Scale bar, 200 μm.

Fig. 5. Delivery of LNP-mRNA to the brain via intracerebroventricular (ICV) injection.

(A) Schematic of stereotactic ICV injection of LNP-mRNA (Cre) in an SRM-1 piglet. (B) Whole-brain coronal sectioning and IHC for tdTomato and NIS was performed to identify mRNA uptake (left panel). Cell transfection was restricted to the outer cortex with minimal penetration into tissue. Three distinct cortical cell types of the brain were identified by morphology and staining differences (arrows, right panel).