Targeted delivery of nucleic acid formulations for in vivo CAR-Treg generation

In this Phase I SBIR, Optimeos will develop an off-the-shelf, in situ CAR-Treg therapy through the nanoparticle-mediated delivery of a CAR-FoxP3 construct to CD4+ T cells as a treatment for transplant rejection. We envision an off-the-shelf therapy that reduces the healthcare costs and high burden of adoptive cell therapies. Ex vivo Chimeric Antigen Receptor Regulatory T cells (CAR-Treg) are currently under development for transplant rejection and other autoimmune diseases, building on the dramatic clinical benefits in cancer from CAR-T effector cells. The first ex vivo CAR-Treg has now moved into the clinic in the STEADFAST trial for kidney transplant patients. CAR-Treg cells directed against the HLA antigen could provide immune tolerance after transplantation without global immune suppression. This will decrease the incidence or severity of transplant rejection in well- matched transplants, and allow more transplants due to less stringent HLA matching requirements. We propose generation of CAR-Treg cells in situ to overcome the complex manufacturing of current approaches. Ex vivo CAR-T cells, both regulatory and effector, require labor-intensive, weeks-long processes starting from the patient’s own cells. These therapies can cost $400,000 or more, which will prove prohibitive for many non-oncologic diseases. Our off-the-shelf CAR-Treg therapy will use nanoparticle-encapsulated mRNA encoding both the CAR, to induce antigen specificity, and FoxP3, to drive the regulatory phenotype. We will employ anti-CD4 antibodies as our targeting moiety to reach the desired population after IV administration. Our Coated Inverse NanoCarriers (CINCs) encapsulate mRNA in a polymer/lipid hybrid structure that minimizes inflammation and enhances targeted delivery by providing stable attachment of the targeting ligand. The targeting capabilities of CINCs will allow us to reach peripheral, splenic, and lymphatic T cells while minimizing non-specific liver clearance. The immune-silent CINC platform permits repeat dosing and minimizes inflammation, a profile that will be essential for in situ CAR-Treg. Over two aims in this proposal, we will select our lead CINC formulation and design the mRNA cargo to generate CAR-Treg cells in vivo. Specific Aim 1: Target CINCs to CD4+ T cells in vivo. We will select the CINC formulation that exhibits both robust transfection of primary human T cells in vitro and high specificity in vivo. We expect to achieve 15x enhancement in delivery to peripheral CD4+ T cells with a >3x reduction in liver delivery. Specific Aim 2: Create and validate mRNA constructs for CAR-Treg generation. Our mRNA sequence design will employ a CAR for HLA-A2 with FoxP3 on the same construct to ensure antigen-specific suppressive function. After in vitro functional validation, we expect a humanized mouse study conducted with our lead CINC formulation to show an expression peak of 5% CAR+ cells within the CD4+ compartment. In Phase II, we plan to demonstrate efficacy in a skin transplant model, bringing a safe, off-the-shelf therapy closer to addressing diseases that affect nearly 50 million Americans.