Encyclopedia
The field of mRNA has made significant progress in the last ten years and has emerged as a highly attractive means of encoding and producing any protein of interest in vivo. Through the natural role of mRNA as a transient carrier of genetic information for translation into proteins, in vivo expression of mRNA-encoded antibodies offer many advantages over recombinantly produced antibodies.
- mRNA
- antibody
- immunotherapy
1. Introduction
Antibodies are components of the human adaptive immune system that are crucial to prevent, control and resolve infections [1]. Prior to the introduction of antibiotics and vaccines, passive transfer of serum containing antibodies from convalescent individuals or animals was the “standard of care” against infectious diseases [2]; this approach is still used today to treat venomous snake bites, toxin exposure, rabies and, more recently, the Ebola virus and SARS-CoV-2 infections [3][4][5][3,4,5]. Over the last two decades, the field of antibody-mediated immunotherapy has been transformed by the development of methods to immortalize B cells [6]. Further breakthroughs in recombinant antibody technologies, such as antibody isolation and gene sequencing, have resulted in the regulatory approval and commercialization of over 100 monoclonal antibodies (mAbs) to treat autoimmune diseases, neurodegenerative disorders, cancer and infectious disease [7][8][9][10][7,8,9,10]. To date, all licensed mAbs are purified IgG proteins that are administered intravenously (IV), intramuscularly (IM) or subcutaneously (SC) [2][8][2,8].
Antibodies are hetero-tetrameric proteins formed by two full-length heavy (H) and light (L) chains held together via charge–charge interactions and disulfide bonds. Two distinct parts of an antibody are critical for its function: the antigen binding fragment (Fab) and the crystallizable fragment (Fc) (Figure 1). The Fab region determines antibody specificity and is composed of one constant and one variable domain (Fv) of the H and L chains. The Fc region, comprised of the constant domains of two H chains, determines the in vivo antibody half-life by binding to the neonatal Fc receptor (FcRn) [11][12] [11,12] and can modulate immune cell activity through binding to Fc receptors on innate immune cells [13]. The Fc effector functions can be altered by post-translational modifications such as glycosylation, methionine oxidation or deamidation, which can also impact antibody distribution and stability [14][15][16][14,15,16].
Figure 1. Schematic diagram of mRNA and antibody designs used in recent studies. (A) Basic structure of an mRNA construct; (B) different formats used by recent studies to encode a full-length antibody as mRNA; (C) derivatives of full-sized antibodies that are comprised of only the heavy chain; (D) bispecific antibody, single-chain variable fragment (scFv) format; SP, signal peptide; VH, variable heavy chain domain; CH, constant heavy chain domain; VL, variable light chain domain; CL, constant light chain domain; Furin-T2A, furin and thosea asigna virus 2A peptide; IRES, internal ribosomal entry site; GPI, glycosylphosphatidylinositol membrane anchor; VHH, VH domain, heavy chain only.
2. mRNA as a Platform for Efficient Protein
Expression In VivomRNA synthesis starts with in vitro transcription (IVT) by RNA polymerases of a linear DNA template that contains a promoter, 5’ and 3′ untranslated regions (UTRs), and an open reading frame [17][53]. Two additional elements are required for the mRNA to be biologically active: an inverted triphosphate cap at the 5′ end and a poly(A) tail at the 3′ end. Both a cap and poly(A) tail are required for efficient translation and stabilization of the mRNA, and both elements can be added either during transcription or enzymatically after [18][54]. Importantly, cap structure [19] [55] and poly(A) tail lengths [20] [56] can impact the amount of protein produced by an mRNA-based therapy (Figure 1A). Other factors that can affect mRNA translation or half-life include UTRs ][21][22][23] [57,58,59] and codon optimization [24][60]; the work of understanding and balancing these mRNA elements is an area of intense research.Protein expression from exogenous mRNA was demonstrated in vivo two decades ago when direct injection of mRNA into mouse muscle was shown to result in local protein expression [25][61]; however, there are still several hurdles that need to be solved. First and foremost, mRNA is an unstable molecule that is degradable by ribonucleases in the body resulting in a very short half-life. Additionally, naked mRNA is not efficiently delivered to cells and mRNA can activate innate immune pathways that may decrease protein expression [26][52]. In the last decade, significant progress has been made in all these areas.
3. Modified mRNA
mRNA and the side products of the mRNA in vitro transcription (IVT) process can trigger an innate immune response through pathogen-associated molecular pattern (PAMP) receptors, such as toll-like receptor (TLR)3, TLR7, TLR8, retinoic acid-inducible gene 1 (RIG-I) and nucleotide-binding and oligomerization domain containing protein 2-(NOD-2), which recognize either double-or single-stranded RNA [26][52]. Activation of the innate immune pathways could interfere with therapeutic applications by decreasing protein expression and undermining tolerability. Modified bases found in natural RNAs have been revealed to not only suppress recognition by TLRs in vivo but also to increase the stability and translation of mRNA [27][28][42,43]. A common modification is replacement of uridine with pseudouridine, which has been shown to increase mRNA translation and decrease innate immune stimulation [28][29][43,62]. Further reduction in stimulation of innate immunity has been obtained by stringent mRNA purification by high-performance liquid chromatography (HPLC), which can remove the aberrant RNAs created in the IVT reaction [30][31][63,64].
4. Self-Amplifying mRNA
In order to increase protein expression and decrease mRNA dose, some groups have developed a self-amplifying mRNA (SAM) approach based on the alphavirus genome that encodes its own RNA replication machinery [32][33][65,66]. Due to the bipartite division of structural and nonstructural regions in the alphavirus genome, structural genes can be replaced with genes of interest while still retaining the machinery for replicative functions (Figure 2). However, there are significant drawbacks to this approach, largely due to the sheer size of the construct and their intrinsically high innate immune stimulation. To encode the polymerase, replicons start at a size of ~7.6 kb; the addition of the gene of interest results in a large construct that is prone to cleavage and therefore unstable [34][35][36][67,68,69]. As such, large-scale manufacturing, storage and characterization of these constructs are quite complex and expensive and constitute an active area of research in the field [32][35][65,68]. To overcome the difficulties of SAM resulting from size, the replicase can be provided in trans [37][70]. While this results in shorter mRNAs, it requires manufacturing of at least two RNAs and efficient delivery of both into the same cell. Another disadvantage of SAMs is their intrinsic immunogenicity due to the formation of short double-stranded RNA during production and self-amplification [35][68]. There will need to be significant improvements in the stability of long mRNAs, immunogenicity and more complex manufacturing before SAM can become a realistic option in the clinic to produce monoclonal antibodies in vivo.
Figure 2. Diagram of the basic structure of self-amplifying mRNA (RepRNA) that includes the typical cap, 5′ and 3′ UTRs and poly(A) tail. The details of the open reading frame are depicted and contain the nsp1, nsp2, nsp3 and nsp4 genes from the alphavirus genome that encode for RNA replication machinery. Downstream from the rep genes are a subgenomic promoter and the elements encoding a monoclonal antibody (mAb). Abbreviations: m7G: 7-methylguanosine; UTR: untranslated region; nsp: nonstructural protein.
