MicroRNAs Enable mRNA Therapeutics to Selectively Program Cancer Cells to Self-Destruct

Abstract

The advent of therapeutic mRNAs significantly increases the possibilities of protein-based biologics beyond those that can be synthesized by recombinant technologies (eg, monoclonal antibodies, extracellular enzymes, and cytokines). In addition to their application in the areas of vaccine development, immune-oncology, and protein replacement therapies, one exciting possibility is to use therapeutic mRNAs to program undesired, diseased cells to synthesize a toxic intracellular protein, causing cells to self-destruct. For this approach to work, however, methods are needed to limit toxic protein expression to the intended cell type. Here, we show that inclusion of microRNA target sites in therapeutic mRNAs encoding apoptotic proteins, Caspase or PUMA, can prevent their expression in healthy hepatocytes while triggering apoptosis in hepatocellular carcinoma cells.

Keywords: RNA modifications; mRNA; miRNA; suicide therapy; therapeutic.

FIG. 1.

Nonspecific biodistribution of protein expression after mRNA delivery. (a) Intravenous delivery of mRNA results in protein expression in multiple tissues. Total flux (photons/s) from indicated organs 6 h after 1-methyl-pseudouridine (m¹Ψ) Luc mRNA encapsulated in KC2 or MC3 LNPs or in complex with L2000 is intravenously administered to mice. (b) Intratumoral delivery of mRNA can lead to protein expression in liver. Total flux (photons/s) from tumor and liver 6 h after m¹Ψ Luc mRNA encapsulated in MC3 LNP is administered to mice with subcutaneous MC38 tumor. (c) Schematic representation to illustrate the possible benefits of using a miRts in mRNAs to dampen off-target expression in mice. Intratumoral injection of an mRNA encoding a toxic protein can trigger cytotoxicity to kill tumor cells but may also damage a nontargeted tissue such as liver. miRts incorporation in the mRNA may restrict expression of toxic proteins to tumor only. Luc, luciferase; LNP, lipid nanoparticle; miRt, microRNA target site.

FIG. 2.

Endogenous miRNAs can be utilized to suppress protein expression from synthetic modified mRNAs in specific cells. (a) Schematic representation of Luc mRNA with a miRts in the 3′ UTR. (b–d) miRts incorporation in modified mRNA leads to suppression of encoded protein in specific cells. RLUs 6 h after L2000-mediated transfection of 1-methyl-pseudouridine (m¹Ψ) mRNA encoding Luc in (b) HeLa, (c) primary human hepatocytes, and (d) RAW 264.7 macrophages. Each datum in the bar graph is an average of three biological samples, and the error bars represent standard deviation. Luminescence from cells with miRts-containing mRNAs was compared with cells with CTRL mRNA, and P values were generated by Prism using the unpaired, two-tailed t-test. ***P < 0.001, ****P < 0.0001. miRNA, microRNA; RLU, relative luminescence units; UTR, untranslated region.

FIG. 3.

Endogenous miRNAs can act as siRNAs on synthetic modified mRNAs with a miR target site. (a) Schematic representation of Luc mRNA with a miRts in the 3′ UTR. (b, c) Endogenous miRNAs can act as siRNAs on modified mRNA and mediate mRNA cleavage. (b) RLUs 24 h after Jurkat cells were EP with 1-methyl-pseudouridine (m¹Ψ) mRNA encoding Luc. Luminescence from cells with 142ts-containing mRNAs was compared with cells with CTRL mRNA, and P values were generated by Prism using the unpaired, two-tailed t-test. ****P < 0.0001. (c) Luc transcript levels normalized to HPRT transcript at various time points after EP. Luc transcript was assessed using three different primer pairs (Pf1-Pr1, Pf2-Pr2, and Pf3-Pr3) depicted in the schematic representation in (a). All values were normalized to Luc relative to HPRT transcript 5 min after electroporation. Bar graph showing percent total reads obtained across the Luc transcript after 5′-phosphate sequencing from total RNA extracted from cells 30 min post-EP. A zoomed-in view of the boxed region in the transcript is shown in the inset. miR142 sequence is shown in blue. (d) Schematic representation of Epo encoding mRNA with a miRts in the 3′ UTR. (e) Endogenous miRNAs trigger mRNA decay and protein suppression for modified mRNA for multiple open reading frames. Serum Epo levels 24 h after Jurkat cells were EP with m¹Ψ mRNA encoding Epo. Epo expression from cells with 142ts-containing mRNAs was compared with cells with CTRL mRNA, and P values were generated by Prism using the unpaired, two-tailed t-test. **P < 0.01. Epo transcript levels normalized to HPRT at various time points after EP. Epo transcript was assessed using the primer pair (Pf-Pr) depicted in the schematic representation in (d). All values were normalized to Epo relative to HPRT transcript 5 min after electroporation. HPRT, Hypoxanthine phosphoribosyltransferase; siRNA, small interfering RNA; EP, electroporated; Epo, erythropoietin.

FIG. 4.

miRNAs can suppress off-target expression in mammals. (a) Schematic representation of mRNA with a miRts in the 3′ UTR. (b–d) miRts incorporation in mRNA enables protein suppression in rodents upon mRNA delivery. Serum Epo levels 6 h after MC3-encapsulated or L2000-complexed 1-methyl-pseudouridine (m¹Ψ) mRNA encoding Epo was administered intravenously to (b) mice or (c) rats. (d) Total flux (photons/s) and serum Epo levels 6 h after MC3-encapsulated m¹Ψ mRNAs encoding Luc and Epo were administered intravenously to “chimeric” mice (mouse hepatocytes replaced with human hepatocytes). (e) miRts incorporation in mRNA enables protein suppression in nonhuman primates upon mRNA delivery. Serum Epo levels 6 h after MC3-encapsulated m¹Ψ mRNA encoding Epo was administered intravenously to African green monkey. (f) Schematic representation of an mRNA with two different miRts's in the 3′ UTR. (g) Incorporation of different miRts's in mRNA enables protein suppression in multiple tissues upon mRNA delivery. Total flux (photons/s) from liver and spleen 6 h after MC3-encapsulated m¹Ψ mRNA encoding Luc was intravenously administered to mice. Error bars represent standard deviation in the figure. Luminescence/Epo expression generated upon intravenous administration of miRts-containing mRNA was compared with levels with CTRL mRNA, and P values were generated by Prism using the unpaired, two-tailed t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

FIG. 5.

Intratumoral delivery of MC3-encapsulated PUMA-122ts triggers apoptosis in tumor cells while alleviating liver toxicity. (a) Schematic representation of PUMA mRNA with miR122 target site(s) (122ts or 3 × 122ts) in the 3′ UTR. (b) Schematic representation of PUMA-mediated apoptosis pathway. (c) PUMA-122ts triggers apoptosis in tumor cells while alleviating liver toxicity in a Hep3b subcutaneous xenograft mouse model. Representative images from tumor and liver IHC for CC3, and H&E staining 6 h after intratumoral injection of 6.25-μg PUMA mRNA. Quantification of percentage CC3-positive area in tumor and liver, and ALT and AST levels in serum are shown. NST represents an RNA with a similar sequence where all identifiable start codons AUG, CUG, and GUG have been removed. Error bars represent standard deviation in the figure. Levels generated upon administration of LNP-encapsulated mRNA were compared with levels with buffer, and P values were generated by Prism using one-way analysis of variance. ns P > 0.05, *P < 0.05, ***P < 0.001, ****P < 0.0001. IHC, immunohistochemistry; CC3, cleaved caspase 3; H&E, hematoxylin and eosin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; NST, nonstart RNA.

FIG. 6.

miR122 target site incorporation alleviates liver damage from systemic delivery of MC3-encapsulated Caspase mRNA. (a) Schematic representation of Caspase mRNA with miR122 target site(s) (122ts or 3 × 122ts) in the 3′ UTR. (b) Intravenous delivery of MC3-encapsulated Caspase-122ts alleviates liver damage from Caspase mRNA. Serum levels of ALT and AST 6 h after intravenous administration of MC3-encapsulated Caspase mRNA in mice. Error bars represent standard deviation in the figure. Representative images from liver IHC for CC3, and H&E staining. NST represents an RNA with a similar sequence where all identifiable start codons AUG, CUG, and GUG have been removed (NST). The P values are not reported in this experiment because of the lower n. The mean and standard deviation for each group are shown in the table.