Therapeutic immunomodulation by rationally designed nucleic acids and nucleic acid nanoparticles

Abstract

The immune system has evolved to defend organisms against exogenous threats such as viruses, bacteria, fungi, and parasites by distinguishing between "self" and "non-self". In addition, it guards us against other diseases, such as cancer, by detecting and responding to transformed and senescent cells. However, for survival and propagation, the altered cells and invading pathogens often employ a wide range of mechanisms to avoid, inhibit, or manipulate the immunorecognition. As such, the development of new modes of therapeutic intervention to augment protective and prevent harmful immune responses is desirable. Nucleic acids are biopolymers essential for all forms of life and, therefore, delineating the complex defensive mechanisms developed against non-self nucleic acids can offer an exciting avenue for future biomedicine. Nucleic acid technologies have already established numerous approaches in therapy and biotechnology; recently, rationally designed nucleic acids nanoparticles (NANPs) with regulated physiochemical properties and biological activities has expanded our repertoire of therapeutic options. When compared to conventional therapeutic nucleic acids (TNAs), NANP technologies can be rendered more beneficial for synchronized delivery of multiple TNAs with defined stabilities, immunological profiles, and therapeutic functions. This review highlights several recent advances and possible future directions of TNA and NANP technologies that are under development for controlled immunomodulation.

Keywords: DAMP; NANPs; PAMP; PRR; immunomodulation; innate immune system; therapy.

Figure 1

Brief overview of cellular innate immunity with an emphasis on nucleic acid recognition. The first line of nucleic acid PRRs consists of TLRs that can sense different PAMPs specific for non-self nucleic acids. Then cytosolic pathogen-associated nucleic acids can be sensed by members of the RLR family (RIG-I, MDA5). The endogenous and viral DNAs can also lead to RIG-I activation following their transcription by cytosolic RNA pol III, or can be detected directly as dsDNAs via cytosolic DNA sensing systems such as the cGAS-cGAMP-STING pathway. All of these pathways initiate the translocation of transcription factors including IRF3/7 and NF-κB to the nucleus and the subsequent induction type I IFN and pro-inflammatory cytokine production.

Figure 2

During contact with stromal fibroblasts, breast cancer cells activate NOTCH1/MYC signaling that leads to higher transcription of ncRNA RN7SL1 carrying 5’-ppp. These transcripts then remain unshielded since levels of their protein-binding partner (SRP9/14) remain constant. Naked RN7SL1 is loaded to exosomes and, upon interaction with breast cancer cells, can activate RIG-I signaling leading to an inflammatory tumor microenvironment that can promote tumor progression and poor clinical outcomes.

Figure 3

The use of endogenous RN7SL1 ncRNA to improve CAR-T cell therapy efficacy. Engineered CAR-T cells transcribe transgenic RN7SL1 ncRNA together with a chimeric antigen receptor. The resulting cell-autonomous effect prevents T-cell exhaustion and increases cell expansion. In addition, excreted exosomes transport RN7SL1 to intratumor myeloid cells, such as dendritic cells, rather than cancer cells, thereby avoiding inflammation triggered by tumor cells.

Figure 4

Aptamers are nucleic acids selected to specifically bind the molecules of interest in a similar manner to monoclonal antibodies. (A) Nanotechnology offers significant advantages in fusing individual aptamers to multivalent or bispecific molecules. Thus, by linking together the same or different aptamers, the increased binding affinity and/or ability to crosslink target cell receptors can be achieved. (B) Bispecific aptamers can promote cell-to-cell interactions with potential immunomodulatory applications. For example, a single stranded bispecific aptamer targeting CD28 on T cells and Multidrug-Resistant-associated Protein 1 (MRP1), involved in chemotherapy on B16 melanoma cancer cells, has been used to provide the necessary co-stimulatory signal for T cell activation. (C) Instead of cell membrane receptors that may be quickly internalized, an alternative strategy could be to target co-stimulatory signals to proteins (e.g., VEGF) overexpressed on tumor stroma.

Figure 5

Schematic depiction of various NANPs, their production, characterization, storage, and handling. (A) Computational 3D visualization of individual NANPs with corresponding representative AFM images. (B) Two orthogonal NANPs design strategies are based either on the presence of both intra- and intermolecular or only intermolecular bonds, which also determine the assembly protocol of corresponding NANPs. (C) Several protocols for efficient one-pot NANPs self-assembly. Protocol (i) promotes secondary structure formation of individual monomers needed for NANPs assembly via long-range interacting motifs. For this assembly protocol, the individual ssRNAs are first denatured by heating at 95°C and then snap cooled on ice to form intramolecular Watson-Crick (W-C) bonds. The following incubation at 30°C in the presence of Mg²⁺ ions allows intermolecular bindings of monomers and assembly of NANPs. In (ii)> protocol, monomers form only intermolecular canonical Watson-Crick base pairs, thus no pre-folding is needed, and any intramolecular interactions should be avoided by design. The (iii) protocol allows for co-transcriptional assembly of different types of NANPs formed as their RNA strands are transcribed from dsDNA templates. (D) Assembled NANPs can be stored and transported in anhydrous forms at ambient temperatures. The impact on structure stability, immunorecognition, and functionality depends of dehydration protocol and needs to be checked after rehydration for each type of NANP.

Figure 6

Most common innate pathways shown to be activated upon NANPs internalization. (A) Intracellular delivery of NANPs requires carriers; naked NANPs are immunoquiescent due to their ineffective crossing of biological membranes. Delivery of RNA rings and cubes trigger the immune system through TLR7. By-passing of TLR sensing can be compensated by RIG-I that can detect RNA NANPs bearing 5’ triphosphates. DNA containing NANPs can be sensed after promoter independent transcription of NANPs strands by RNA pol III. DNA fibers stimulate cellular immunity through cGAS-cGAMP-STING pathway. Additionally, interdependent DNA/RNA fiber NANPs can be rationally designed to release of RNAi inducers and NF-κB decoys upon their intracellular re-association. This results in gene specific silencing while simultaneously blocking NF-kB translocation to nucleus thus lowering the proinflammatory immune responses. (B) Some of the architectural and compositional parameters that define immunorecognition of NANPs.

Figure 7

Distribution of FDA approved noncoding nucleic acid therapeutics according to type (A) and route of administration (B). ASO- antisense oligonucleotides, siRNAs- small interfering RNAs, APTs- aptamers.