Targeted Delivery of mRNA Therapeutics—4 Recent Examples
TriLink BioTechnologies, part of Maravai LifeSciences
Delivery of therapeutic agents to specific cells of interest, i.e., “target cells”, is widely regarded as the “holy grail” of drug development regardless of the drug’s molecular size or mechanism of action. For mRNA therapeutic agents, which are unstable toward RNases and are not efficiently taken up by cells, encapsulation or complexation in nanoparticles is generally used for delivery. This blog discusses the following recent examples of targeting moieties for lipid nanoparticle (LNP) delivery of mRNA to specific cells of interest as possible therapeutics:
These examples use mRNA catalog and custom synthesis products available from TriLink.?
1. Monoclonal Antibodies
Antibody-based targeting is attractive because of the high specificity of a monoclonal antibody (mAb) binding to its corresponding antigen and the ability to obtain mAbs for virtually any antigen of interest. Recently, a strategy potentially applicable to many combinations of mRNAs and mAbs was reported by?Rosenblum et al.?(2020). The approach was exemplified with?TriLink’s?CleanCap? Cas9 mRNA?and a mAb for epidermal growth factor receptor (EGFR), which is commonly upregulated in cancer cells.?
As generalized in?Figure 1, LNPs containing a mRNA of interest were coated with cell-targeting mAbs (red) via a novel membrane-anchored recombinant lipoprotein (blue). The lipoprotein was incorporated into the LNP and interacted with the mAb Fc-domain. Because the lipoprotein can bind to the Fc-domain of any mAb, the approach is broadly applicable to target a range of mRNAs to any cell-surface marker for which a cell-targeting mAb is available.?
To explore the potential of therapeutic CRISPR-based genome editing in cancer using this method, Rosenblum et al. studied two cancer cell lines: murine glioblastoma (GBM) and drug-resistant, metastatic human ovarian (OV) cancer.?In vitro?treatment of each cell line with anti-EGFR mAb anchored to LNPs containing CleanCap? Cas9 mRNA and a single-guide RNA (sgRNA) against the proto-oncogene polo-like kinase 1 (PLK1) led to 91% GBM and 84% OV genomic editing, cell-cycle arrest and, ultimately, cell death.?
In vivo?treatment of each cell line was then studied. A single local intracerebral injection into mouse-brain implanted GBM enabled up to ~70% gene editing, causing tumor cell apoptosis, inhibition of tumor growth, and 30% improved survival. For disseminated OV tumors in mice, a single intraperitoneal injection of the EGFR-targeted Cas9 mRNA-PLK1 sgRNA-LNPs led to ~80% gene editing, inhibition of tumor growth, and increased survival by 80%.?
2. Synthetic Peptides?
The following are two different mRNA delivery approaches using peptides that are readily available by means of conventional synthesis methodologies.
Receptor Targeting:?Hepatocellular carcinoma (HCC)—the most prevalent liver cancer—has a high mortality rate and dismal prognosis. Enhancing anti-tumor immunity using the immune checkpoint inhibitor?anti-PD-L1 (aPD-L1) mAb?has shown promise, but only in a minority of HCC patients, due to mutational absence of?p53 tumor suppressor?gene activity.?
To achieve HCC targeting of LNPs containing functional, non-mutated p53 mRNA,?Xiao et al.?(2022) used a synthetic peptide ligand, termed CTCE, which specifically binds to CXCR4, a receptor that is upregulated in HCC. Following is a brief summary of key results they obtained using?TriLink custom synthesized CleanCap? p53 mRNA (pseudouridine and 5-methylcytosine substituted), in conjunction with TriLink reporter mRNAs encoding either Cy5-labeled firefly luciferase (Cy5-Luc) or enhanced green fluorescent protein (EGFP) mRNA.
The CTCE peptide was covalently linked to one of the LNP lipid components containing EGFP mRNA for optimization of LNP CTCE-surface density, which was quantified by FACS measurements following incubation with HCC cells?in vitro. Next, mice with grafted HCC tumors received tail vein (i.v.) injections of Cy5-Luc mRNA in CTCE-LNPs, which led to significant intratumoral accumulation of this labeled reporter mRNA.?
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To examine the role of p53 in immunosuppression of HCC, Xiao et al. then studied the effects of administering p53 mRNA formulated in CTCE-LNPs and aPD-L1 mAb to mice with grafted p53-null HCC?tumors. Mice received i.v. injections of either p53 mRNA formulated in CTCE-LNPs, or aPD-L1 mAb, or a combination of both the mRNA and mAb every 3 days for 4 total injections, with frequent/periodic tumor growth monitoring by ultrasound imaging. The combination treatment led to ~2-fold reduction in tumor growth and ~2-fold longer survival compared to either treatment alone, with no evidence of adverse effects.?
Based on these findings, targeted co-administration of p53 mRNA (formulated in CTCE-LNPs) with aPD-L1 mAb therapy could provide an improved treatment strategy for p53-deficient HCC and potentially other p53-deficient cancers.
Cell-Penetrating Peptides (CPPs):?Although facilitated cellular uptake of CPP-drug conjugates has been extensively investigated,?Porosk et al.?(2023) note that CPP-LNP delivery of mRNA is relatively unexplored. Therefore, they investigated using various LNPs comprised of lipidated CPP conjugates for the delivery of?CleanCap? 5-methoxyuridine-substituted Luc mRNA?in mice. After a single injection,?Luc reporter expression levels were evaluated using either whole tissue homogenates postmortem or bioluminescence live-animal imaging. One of the tested CPPs led to near exclusive (~95%) splenic expression of Luc, primarily in antigen-presenting dendritic cells. This finding supports the possibility of a cancer immunotherapy strategy wherein a tumor antigen-encoding mRNAs in CPP-LNPs are targeted to antigen-presenting dendritic cells to activate cytotoxic T cells.?
3. Synthetic Lipid Headgroups?????????
In contrast to the above-mentioned mRNA-LNP targeting approaches using mAB/antigen and ligand/receptor binding specificity,?Wang et al.?(2022) developed a novel screening method to discover new LNP compositions that allow for selective organ targeting, and termed it “SORT”. A detailed step-by-step methodology for SORT was published at the end of 2022 and as of April 2023, it has been accessed more than 19,000 times, indicating a high level of interest in the scientific community. The reporter protein used for optimizing SORT was?TriLink’s CleanCap? 5-methoxyuridine-substituted Luc mRNA.?
The strategy for SORT derives from the fact that lipids in LNPs are comprised of long hydrocarbon chains that, on one end, have a so-called “headgroup” that is exposed on the LNP’s outer surface. Headgroup-mediated binding to external molecules—primarily proteins—leads to a biomolecular coating called a “corona” (Kopac?2020). In SORT, the chemical nature of lipid headgroups in LNP formulations is varied to empirically find headgroups leading to altered cell-type affinity.?
The method starts with a LNP mixture comprised of three types of lipids (cationic, ionizable, and PEGylated) and cholesterol, all in a fixed ratio. To this LNP mixture a SORT test-lipid (X) is added as a fifth component over a range of values (v). The numbers for X and v thus determine the size of the experimental matrix for testing. To test the LNP formulations in vivo, they were i.v. administered to mice for measurement of Luc expression in various excised organ tissues of interest. Wang et al. provide exemplary specificity data for three SORT lipid headgroups: trimethylammonium cation for liver, phosphate anion for spleen, and dimethylamino for lung.?
The scientists are screening an expanded set of lipids and SORT lipid headgroups for additional novel compositions that show selectivity for tissue types beyond liver, spleen, and lung.?
4. Fibronectin Mimicking??
The feasibility of targeted delivery of mRNA-LNPs to solid tumors was studied by?Qin et al.?(2022). Tumor cells biochemically communicate with their extracellular matrix (ECM) protein fibronectin through overexpressed integrin receptors on their cell surfaces. Consequently, Qin et al. considered the fibronectin binding domain for integrin (Arg-Gly-Asp, RGD) to be a potential targeting moiety for mRNA delivery to solid tumor cells.
Twenty synthetic RGD-conjugated ionizable lipids were combined with a mixture of traditional lipid (DOPE), PEGylated lipid, and cholesterol to obtain 20 unique LNP formulations loaded with Luc mRNA reporter. A human HCC cell line (Hep2G) was used for screening each RGD-LNP vs. an untargeted LNP control. This led to the identification of a top performer, termed 1A-RGD-LNP, which showed ~2-fold higher Luc expression relative to the control. This enhanced Luc expression was abrogated by pretreatment of Hep2 cells with soluble RGD peptide, consistent with 1A-RGD-LNP/integrin-mediated delivery.?
Next, the researchers studied the feasibility of tumor-targeted CRISPR gene editing with co-delivery of?TriLink Cas9 mRNA?and EGFP-targeted sgRNA formulated in 1A-RGD-LNPs to EGFP+-Hep2G cells?in vitro. After a 7-day incubation period, flow cytometry measurements showed up to 90% knockout of EGFP expression compared to untreated control.?
Concluding Comments
Many more examples of the above lines of research, and other approaches, are discussed in a recent review by?Meyer et al.?(2022) titled?Targeting Strategies for mRNA Delivery. This Open Access article, written by a team of biopharmaceuticals experts at AstraZeneca and Johns Hopkins University School of Medicine, is a comprehensive resource with 190 references. As such, it provides a very useful discussion of this exciting aspect of mRNA research for further reading.?