There are several types of RNA-based therapies and vaccines including messenger or mRNAs, small interfering RNAs (siRNAs), microRNAs (miRs) and antisense oligos (ASOs)1. The most well-known RNA-based modalities are the COVID-19 vaccines that contain the mRNA coding for the SARS-CoV2 spike protein encapsulated in lipid nanoparticles (LNPs). The rapid development and approval of the COVID-19 mRNA vaccines have highlighted the immediate need for robust toxicology programs for RNA-based therapies and vaccines, so comprehensive toxicology programs are actively being developed and implemented. RNA-based therapies can be formulated in a few ways – in some cases, naked mRNA is administered directly in a buffer but most RNA based drugs and vaccines are combined with lipid-based nanoparticles of LNPs. Additionally, several oligonucleotide modifications using complex chemistry have been developed to improve delivery efficiency and decrease the RNA degradation2. It is important to consider the formulation when designing tox studies and vaccines and targeted therapies pose different efficacy and toxicology challenges.
In the case of vaccines, organ-specific inflammation has been reported along with a small number of long-term adverse effects and acute adverse effects. Prior to the approval of the COVID-19 vaccine, non-GLP toxicity studies in rats showed specific toxicities that could be explained scientifically3. For example, increased lymphocyte counts were attributed to the expected immune response, while hepatocyte vacuolation was attributed to LNP uptake in the liver3. Due to the fact that acceptable toxicities were detected, the COVID-19 vaccines were deemed as safe and tolerable for systemic administration to humans to induce a broad immune response.
In contrast, targeted therapies that are systemically dosed have issues with tissue specific tropism and delivery efficiency. Typically, mRNA-LNP therapies are administered locally such as direct injection into the eye or injection into the cerebrospinal fluid (CSF) for delivery to the CNS2. When administered systemically into the bloodstream, mRNA therapies tend to accumulate in the highly vascularized liver, thus reducing the amount of therapy to the target organ while increasing the risk of liver toxicity. Additionally, kidney toxicity needs to be monitored since the RNA-LNP complexes are cleared through renal filtration. Interestingly, specific toxic effects have been identified due to RNA-based therapies including activation of the immune response4. Binding of oligonucleotide complexes to serum proteins or cell surface proteins can trigger vascular and intracellular toxicity including activation of complement cascade and development of thrombocytopenia4. Additionally, nucleotide metabolites generated through the partial degradation of oligonucleotides have been reported to inhibit cell proliferation in CNS tissues4.
Since most drug developers have focused on the discovery aspects of RNA-based therapies, there is limited guidance on preclinical safety assessment for RNA-based therapies. In 2023, a group based in EU, DARTER (Delivery of Antisense RNA Therapeutics) published a framework for toxicology assessments of oligonucleotide-based therapies5. This group recommends that drug developers consider preclinical safety testing for different oligonucleotide therapies early in the discovery phase. Toxicity caused by oligonucleotide therapies can be broadly segmented into 3 groups – hybridization and sequence dependent, hybridization dependent and backbone dependent5. Hybridization dependent toxicity can be further divided into on- and off-target effects of the oligonucleotide therapy. Examples of on-target toxicity include excessive efficacy in the target tissue or unwanted tissue tropism. For example, hepatotoxicity and renal toxicity would be classified as off-target toxicity. There have been reports of the immune system activation caused by specific oligonucleotide sequences and some of the well-studied nucleotide motifs have been show to trigger TLR (toll-like receptor) signaling5. While thrombocytopenia has been reported as a toxic response to oligonucleotide therapies, the underlying mechanism remains unclear.
Based on these early reports, it is clear that while RNA-based therapies are relatively rapid and cost-effective to develop, it is important to develop a robust plan to assess toxicity of each therapy based on the sequence, unique chemistry, formulation and delivery route.
References:
1https://www.sciencedirect.com/science/article/abs/pii/S1877117323002041#
2https://www.nature.com/articles/s41573-020-0075-7#Sec9
3https://pmc.ncbi.nlm.nih.gov/articles/PMC9965811/
4https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2022.1006304/full
