Approaches towards biomaterial-mediated gene editing for cancer immunotherapy

Abstract

Gene therapies are transforming treatment modalities for many human diseases and disorders, including those in ophthalmology, oncology, and nephrology. To maximize the clinical efficacy and safety of these treatments, consideration of both delivery materials and cargos is critical. In consideration of the former, a large effort has been placed on transitioning away from potentially immunoreactive and toxic viral delivery mechanisms towards safer and highly tunable nonviral delivery mechanisms, including polymeric, lipid-based, and inorganic carriers. This change of paradigm does not come without obstacles, as efficient non-viral delivery is challenging, particularly to immune cells, and has yet to see clinical translation breakthroughs for gene editing. This mini-review describes notable examples of biomaterial-based gene delivery to immune cells, with emphasis on recent in vivo successes. In consideration of delivery cargos, clustered regularly interspaced palindromic repeat (CRISPR) technology is reviewed and its great promise in the field of immune cell gene editing is described. This mini-review describes how leading non-viral delivery materials and CRISPR technology can be integrated together to advance its clinical potential for therapeutic gene transfer to immune cells to treat cancer.

Figure 1:

Schematic of the primary in vivo non-viral gene delivery barriers associated with common non-viral vehicles. As loaded delivery carriers travel through systemic circulation, they encounter many anatomical barriers, including epithelial/endothelial linings and the extracellular matrix (ECM). Additionally, professional phagocytes in the area are responsible for colloidal clearance, limiting a carrier’s ability to act directly on target cells. Likewise, proteins (i.e. nucleases) exist in the blood and ECM to degrade exposed nucleic acids. Ultimately, crossing the cellular plasma membrane is a key rate limiting step for overall biomaterial gene transfection. Because nucleic acids cannot typically pass through the membrane unprotected, physical means or active cellular uptake mechanisms (endocytosis, pinocytosis, phagocytosis, fusion) are necessary. Intracellular steps of endosomal escape, nucleic acid release from the vehicle, and trafficking/translocation are then necessary for successful transfection.

Figure 2.

Design and in vivo functionality of lymphocyte-programming nanoparticles. (A) Schematic describing the fabrication of the poly(β-amino ester) nanoparticles. (B) Confocal microscopy indicates rapid internalization of particles from the cell surface of T cells within 120 min. (C) Biodistribution of nanoparticles. A bar graph on the right represents percentages of splenocytes positive for fluorescently labelled nanoparticles in animals treated with CD3-targeted nanoparticles. T cells (CD3⁺), macrophages (F4/80⁺, CD11b⁺, CD11c⁻), monocytes (CD11b⁺, Gr1⁺, F4/80^low), and B cells (B220⁺) were measured using flow cytometry. (D) Bioimaging of firefly luciferase expressing leukemia cells systemically injected. Ex vivo CAR-T cells, a current standard, were used for comparison. Reproduced from reference 21 with permission from Springer Nature, copyright 2017.

Figure 3.

CRISPR/Cas9 engineering of T cells in patients with cancer. (A) T cells were isolated from four cancer patients (UPN07, UPN27, UPN35, and UPN39) and were loaded with CRISPR/Cas9 RNPs delivering three sgRNAs that resulted in disruption of the TRAC and TRBC (leading to endogenous TCR deletion) and the PDCD1 (leading to PD-1 deletion) loci. The cells were then transduced with a lentiviral vector to express a TCR specific for the cancer antigen NY-ESO-1. (B) The frequency of TRAC, TRBC, and PDCD1 editing in the total T cell population was determined. (C) Cytoxicity of NYCE T cells (T cells with both CRISPR knockouts and TCR transduction) against NY-ESO-1-expressing cancer cells was measured and compared to T cells with TCR transduction but without CRISPR editing (NY-ESO-1 TCR) and T cells with CRISPR editing but without TCR transduction (CRISPR). (D) The average on-target CRISPR/Cas9 editing efficiency for each target in each patient was measured. (E) To ensure that the NYCE T cells retain their antigen-specific cytolytic activity after infusion, the T cells were recovered from patients at the times specified post-infusion and expanded in the presence of the NY-ESO-1 antigen. The ability of the expanded effector cells to elicit a cytotoxic effect against NY-ESO-1-expressing cells A375 and Nalm-6 NY-ESO-1 compared to non-antigen-specific Nalm-6 cells was measured. Reproduced from reference 84 with permission from American Association for the Advancement of Science, copyright 2020.

Figure 4.

CRISPR/Cas9 nanoparticles for genetic engineering of macrophages for cancer immunotherapy. (A) Nanoparticle-mediated delivery of CRISPR/Cas9 machinery to macrophages to knock down SIRP-⍺, resulting in cancer cell phagocytosis. (B) Schematic of particle design. Cas9 was tagged with a nuclear localization signal (NLS) and an E20-tag. E20-tagged Cas9 and cationic arginine nanoparticles (ArgNPs) were mixed together and self-assembled into superstructures via carboxylate-guanidium binding. The particles are then delivered intracellularly via a membrane fusion mechanism that leads to direct payload release into the cytoplasm. (C) Delivery efficiency of Cas9 to the cytoplasm was assessed by delivering fluorescently labeled Cas9 to RAW264.7 macrophages in vitro (nuclei stained with Hoechst). (D) Delivery of Cas9 RNPs to target the SIRP-α gene in RAW264.7 cells resulted in efficient gene editing, as determined by an indel (insertion and deletion) assay. (E) Fluorescence histogram on Cas9-treated RAW264.7 cells (purple) and untreated cells (green) stained with APC anti-SIRP-⍺ knockout indicates that the knockout of SIRP-⍺ did occur. (F) Percentage of phagocytosis of U2OS cancer cells after co-culture with untreated RAW264.7 cells or SIRP-⍺ knockout cells. Reproduced from reference 94 with permission from American Chemical Society, copyright 2018.