Enhancing mRNA Therapeutics in Two Shakes of a Poly(A) Tail
Keywords: mRNA vaccine, polyadenylation, poly(A) tail, mRNA stability
Recent advances in mRNA-based therapeutics and vaccines show great promise for the future of precision medicine. This mode of delivery enables the expression of a diverse range of proteins, including intracellular, transmembrane, and secreted proteins, without the concerns of genomic integration. Consequently, it has become increasingly popular in therapeutic development for treating a broad range of diseases, including genetic disorders, infectious diseases, and cancer. Notably, this approach was instrumental in the rapid design and production of the first authorized COVID-19 vaccines, demonstrating its potential for flexible and cost-effective therapeutics. However, while mRNA technology has greatly improved in recent years, efforts continue to improve mRNA stability, delivery, and immunogenicity while adhering to safety and GMP regulations. One particular point of study and optimization is the poly(A) or polyA tail, which significantly impacts both stability and translation efficiency. This article will explore how poly(A) tailing is achieved for in vitro transcribed (IVT) mRNA, discuss how optimizing poly(A) tail length improves therapeutic efficiency, and outline VectorBuilder’s quality control strategies to ensure consistent poly(A) tailing for optimal results in both research and clinical settings.
Transcription of mRNA and importance of poly(A) tails
Transcription of mRNA is an intricate process which requires capping, splicing, and polyadenylation of transcripts before they can be translated into functional proteins. These processes occur co-transcriptionally and are regulated by several enzymatic and regulatory proteins, as summarized in Figure 1. Following transcription and processing, an mRNA typically comprises a 5’ cap, a 5’-untranslated region (UTR), a 3’-UTR, an open reading frame encoding the relevant target protein, and a 3’-poly(A) tail. For therapeutic purposes, mRNA is also generally encapsulated within lipid nanoparticles (LNPs) for enhanced mRNA stability and targeting efficiency.
Poly(A) tails are stretches of adenosines typically 70-200 nucleotides in length found at the 3' ends of nearly all eukaryotic mRNAs that play a crucial role in mRNA nuclear export, stability, and translation. The poly(A) tail plays an important role in nuclear export by recruiting factors that mediate the export of mature mRNA through nuclear pores to the cytoplasm. Poly(A) binding proteins (PABPs) play a dual role in regulating mRNA stability: these proteins both protect the poly(A) tail from degradation and have been shown to lead to enhanced targeting of degradation by the exosome. Thus, while it was previously thought that longer poly(A) tails result in enhanced mRNA stability, recent research has indicated that the relationship between poly(A) tail length and mRNA kinetics are incredibly complex and still remain unclear, but current studies indicate that decreased mRNA stability is not associated with its translational efficiency.
While there is no correlation between poly(A) tail length and translation, it has been reported that there are optimal tail lengths at which mRNA transcripts are highly translated. It is hypothesized that significantly shorter (or longer) poly(A) tails hinder (or interfere) with the formation of the closed-loop structure required for translation initiation, resulting in poor translation efficiency. Taken together, studies suggest that poly(A) tail length plays a crucial role in regulating mRNA stability and translation, albeit via independent manners. The exact mechanisms by which these occur bear practical implications both for a better understanding of mRNA biology and advancing mRNA-based therapeutics but have yet to be thoroughly elucidated.
Leveraging the dynamics of polyadenylation and poly(A) tails in translational regulation is key in advancing mRNA-based therapeutics and vaccines in order to manipulate the mRNA life cycle and enhance its therapeutic efficacy.
Figure 1. Mechanism of co-transcriptional polyadenylation in the nucleus. Click on each step to see further information about key components and activities of mRNA processing and transport. Polyadenylation is initiated the nucleus when cleavage and polyadenylation specificity factor (CPSF) recognizes and binds to a highly conserved hexameric polyadenylation signal (PAS) on the pre-mRNA while cleavage stimulatory factor (CstF) binds to a GU/U-rich region (downstream sequence element; DSE); flanking of the cleavage site (predominantly a CA dinucleotide). Along with cleavage factors I and II (CFI, CFII) as well as the poly(A) polymerase (PAP), CPSF and CstF trigger endonucleolytic cleavage. Next, PAP catalyzes addition of adenine residues to the new 3’ end in a template independent manner. Shortly after synthesis of the poly(A) tail begins, nuclear poly(A) binding protein (PABPN) binds to the newly synthesized poly(A) tail and further stimulates polyadenylation by increasing the affinity of PAP for the poly(A). The CPSF-PAP-PABPN complex continues to elongate the tail until it has reached its appropriate length – at which point CPSF separates from the poly(A) polymerase and PABPN leading to a halt in enzymatic activity and termination of polyadenylation. Following export from the nucleus, the unstable binding of PABPN is replaced by cytosolic poly(A) binding protein (PABPC), which in turn binds to eukaryotic initiation factor 4 complex (eIF4G) on the 5’ end of the same mRNA, induces the formation of a closed-loop structure, and subsequently significantly enhances the initiation of translation.
Achieving poly(A) tailing in the laboratory
Polyadenylation of IVT mRNA can be achieved in the laboratory using two different methods (Figure 2): 1) enzymatic polyadenylation or 2) template-encoded polyadenylation.
Enzymatic polyadenylation is carried out following an in vitro transcription reaction by incubating the IVT mRNA with a poly(A) polymerase and ATP, resulting in the addition of adenine residues to the 3’ end of the mRNA. A major advantage of this method is the flexibility to generate poly(A) tails of different lengths, which can be done by modifying the concentration of input RNA, ATP, enzyme, reaction time, or a combination of these. While this approach is great for pilot studies or optimizations, it requires the use of additional enzymes and can therefore significantly increase production costs, especially in large-scale production. Additionally, it is not possible to generate mRNAs with consistent poly(A) tails within a single reaction, leading to high variability in poly(A) tail length.
Figure 2. Comparison of polyadenylation methods used for IVT mRNA.
Template-encoded polyadenylation is accomplished by cloning a stretch of thymines onto the end of the gene of interest encoded by the IVT plasmid. During in vitro transcription using T7 RNA polymerase, template-derived thymines are converted into adenines, resulting in their incorporation at the 3' end of the mRNA. A major advantage of this approach is that it is a one-step reaction, reducing costs and making it more suitable than enzymatic tailing for large-scale production. Additionally, this method enables the generation of poly(A) tails with consistent lengths. However, synthesizing, cloning, and maintaining long stretches of thymines on plasmid DNA can be extremely difficult for a variety of reasons. Primarily, synthesizing and cloning long stretches of repetitive sequences are generally a limiting factor in DNA synthesis. In addition, even upon cloning into the IVT vector and after bacterial transformation, bacterial host strains tend to recombine long stretches of nucleotides, resulting in the loss or degradation of these nucleotides over time. Repetitive sequences also complicate quality control, as traditional methods such as Sanger sequencing lose accuracy.
| Advantages | Disadvantages | |
|---|---|---|
| Enzymatic tailing |
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| Template-encoded tailing |
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Table 1. Summary comparison of poly(A) tailing methodologies.
Optimizing poly(A) tailing for maximum therapeutic efficacy
Due to the consistency and simplified production of poly(A) tails generated by template-encoded tailing, it is typically the preferred method for polyadenylation of IVT RNAs used for research and therapeutic purposes. To overcome the challenges associated with synthesizing and cloning long stretches of adenines, VectorBuilder has optimized the development of IVT mRNA vectors with a variety of poly(A) lengths that can be stably maintained in bacterial host strains and reduced the amount of by-product present in the final product (Figure 3).
Figure 3. Stability assessment of IVT mRNA vectors containing 60-150 nucleotide pure poly(A) tails. Plasmids extracted from large-scale E. coli fermentation were digested with restriction enzymes. The segments containing the poly(A) tail were visualized by polyacrylamide gel electrophoresis. Smearing indicates presence of unstable poly(A) by-product.
While typical mammalian poly(A) tails are around ~200 nucleotides in length, shorter tails are used in research and therapeutic settings as they are easier to produce and offer sufficient stability for translation. We have found that a poly(A) tail length of 100 nucleotides is optimal for maximal protein expression and that further increases in tail length do not enhance translation efficiency (Figure 4).
Figure 4. The influence of poly(A) tail length and structure on translational efficiency. (A-B) HEK293T cells were grown on a 12-well plate and transfected with 1 µg of IVT EGFP mRNA with the same Cap1 and UTRs but different poly(A) tail lengths as shown above. EGFP expression was quantified post-transfection by flow cytometry. Representative images were taken at 24 h and 48 h post-transfection. (C) One-cell stage zebrafish embryos were screened for EGFP expression at 6 h post-microinjection of 250 pg of zebrafish EGFP IVT mRNA.
While this principle can be extended to other IVT mRNA vectors, the optimal poly(A) tail length for specific transcripts should be determined on a case-by-case basis. VectorBuilder offers IVT mRNA vectors with varying poly(A) tail lengths as well as CRO services for tailored optimization of poly(A) length to help researchers and therapeutic developers achieve optimal expression.
Quality control for accurate poly(A) tail length
Quality control of IVT mRNA transcripts, including the assessment of consistent poly(A) tail lengths, is essential to ensure optimal performance. However, the repetitive sequence of a poly(A) tail can pose challenges for traditional methods such as Sanger sequencing and calls for more advanced techniques, such as capillary electrophoresis, liquid chromatography-mass spectrometry (LC-MS), or more sophisticated sequencing modalities such as next-generation sequencing.
While capillary electrophoresis is able to provide high-resolution data on poly(A) tail lengths, this method is unable to detect the presence of variations, mutations, or impurities within the tested poly(A) tail. Utilizing more advanced sequencing methods such as next-generation sequencing (NGS) or Poly(A)-seq also has many limitations. These include requiring a PCR amplification step that can introduce biases and affect accuracy of the final measurement, as well as high complexity, and/or high costs. VectorBuilder utilizes LC-MS to accurately, efficiently, and rapidly determine the molecular weight of poly(A) tails produced in IVT reactions. This high-throughput method provides high-resolution data on poly(A) tail length as well as heterogeneity and is therefore indispensable in IVT mRNA quality control and therapeutic development, as shown in Figure 5.
| Advantages | Disadvantages | |
|---|---|---|
| Liquid chromatography-mass spectrometry (LC-MS) |
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| Capillary electrophoresis |
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| Next-generation sequencing (NGS) |
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Table 2: Comparison of common quality control approaches to analyze poly(A) tail fidelity.
Figure 5. Poly(A) tail size analysis. Poly(A) tails were cleaved from IVT mRNA using ribonuclease T1 and isolated by oligo dT affinity chromatography. Isolated poly(A) tails were analyzed by (A) Urea-PAGE gel electrophoresis and (B) LC-MS. The deconvoluted spectrum was generated from 120 pmol of poly(A) tails with an expected size of 120 nt. (C) Size distribution of poly(A) tails with an expected size of 120 nt. No other transcripts were detected outside this range. Error bars represent standard deviation from triplicates.
Conclusion
Polyadenylation of mRNAs is crucial for stability in the cytoplasm and efficient translation. In cells, polyadenylation is achieved through a highly regulated enzymatic process that occurs co-transcriptionally, whereas in the laboratory, this process has been replicated via two independent methods. While both methods have unique considerations, template-encoded tailing has emerged as the most widely used method due to the consistency of the poly(A) tails produced, lower costs and mutation risk, and compatibility with GMP regulations.
As a result of VectorBuilder’s extensive expertise in vector design and development, we are able to overcome challenges generally associated with template-encoded tailing and produce IVT mRNA transcripts with highly consistent poly(A) tail lengths. The vectors are offered in a variety of poly(A) tail lengths, allowing researchers and drug developers to determine the optimal length for efficient translation of their construct. Our wide range of end-to-end solutions for therapeutic IVT RNA development include untranslated region (UTR), coding sequence, and poly(A) tail length optimization for optimal expression and efficiency, as well as UTR engineering and lipid nanoparticle (LNP) optimization for enhanced targeting. To learn more about how you can partner with VectorBuilder to achieve optimal IVT mRNA design, manufacturing, and extensive quality control, please visit our Therapeutic IVT RNA page.
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