mRNA Gene Delivery Solutions

VectorBuilder provides a one-stop solution for the development of mRNA-based therapeutics, such as vaccines, gene editing, chimeric antigen receptor (CAR), and protein expression in cells or embryos. Based on extensive design and production experience, our team can support researchers for in vitro transcription (IVT) vector design, vector cloning, in vitro mRNA synthesis, mRNA-LNP production, and in vitro/in vivo functional testing to accelerate the development of mRNA-based vaccines and gene therapy.

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Service Details

IVT vector design and cloning
  • IVT vector design based on our backbone optimized for highly efficient in vitro transcription (Figure 1).
  • Variety of in-house validated 5’ & 3’ UTRs and polyA tails (including a 110 bp polyA tail) available for sourcing.
  • Custom IVT vector cloning compatible with downstream linearization, capping, and polyadenylation with rapid turnaround.
  • Codon optimization support through literature-based, computational, and experimental approaches.

Figure 1. A general map of the IVT vector

In vitro mRNA synthesis & lipid nanoparticle (LNP) encapsulation
  • T7 RNA polymerase-based synthesis for conventional and self-amplifying mRNA of up to 10,000 nt from ug to hundreds of mg scale.
  • 5’ capping with cap 1 and 3’ template-derived 110 nt polyA tail for enhancing mRNA stability and translation.
  • Modified nucleotides, such as N1-Methylpseudouridine (m1Ψ) and 5-Methylcytosine (m5C), can be incorporated during synthesis to enhance mRNA translation and immune evasion.
  • Variety of RNA purification options including magnetic beads and chromatography.
  • High-quality mRNA-LNP encapsulation at mg scale.
  • Comprehensive quality controls and LNP profiling.
  • Process development and scale-up for large-scale mRNA manufacturing.
In vitro and in vivo testing of mRNA-based gene delivery
  • Leveraging our high-throughput cloning, production, and testing platforms, we utilize an experiment-oriented strategy to optimize mRNA UTRs, coding sequence, and production methods.
  • We have established functional validation platforms for various applications, such as antigen presentation, antibody expression, CAR expression, and CRISPR.
  • We offer clinically oriented CRO services to assess mRNA-LNP gene delivery efficacy and safety using animal models including rodents and nonhuman primates (NHPs).

Technical Information

Advantages of mRNA-based therapeutics

Using messenger RNA (mRNA) to express therapeutic proteins in humans has been proposed and tested in animal models for decades. Thanks to improved understanding of mRNA biology and recent advances with mRNA in vitro transcription (IVT) and lipid nanoparticle (LNP) technology, several technical hurdles have been resolved, which made the SARS-CoV-2 mRNA vaccines feasible at an unprecedented speed after the outbreak. mRNA possesses unique merits compared to other biologics and is a promising candidate for development and use as a drug.

First, in contrast to recombinant protein drugs, mRNA carried in LNPs can be efficiently delivered through cell membranes and rapidly translated into therapeutic proteins in target cells. Intracellular delivery of therapeutic proteins to cytosol via direct membrane translocation is often limited by membrane impermeability and the nature of the protein, such as charge and molecular weight. Most exocellular proteins enters through endosomal uptake and usually are sequestered in endosomal compartments and degraded by proteases. For successful protein delivery into the cytosol to occur the target proteins often need to be engineered with additional modifications for membrane translocation or endosomal escape. As for mRNA, LNPs can fuse with the cell membrane and release encapsulated mRNA into cytosol. Further, targeted mRNA expression in vivo can be achieved by combining functionalized LNP and optimized mRNA UTRs. Second, unlike many viral vectors that permanently leave foreign DNA in the nucleus or incorporated into the host cell genome, mRNA is degraded by the host cell in days without altering the genome. Viral vector-based vaccines come with the risk of insertional mutagenesis which can have deleterious effects on the cell if integration occurs in an important regulatory or coding region. Thirdly, the production of mRNA can be more cost-efficient and easier to scale up than viral vectors. Since mRNA in vitro transcription occurs in a cell free system, cell-derived impurities and contamination are no longer a concern, in addition, the standardization for process development and large-scale production becomes easier. Altogether, mRNA-LNPs are emerging as promising candidates for vaccine development, protein replacement, genome editing, etc., and VectorBuilder provides the necessary services for their design, production, and optimization.

mRNA production process and QC

Typical workflow of mRNA synthesis and LNP packaging

Figure 2. Typical workflow of mRNA synthesis and LNP packaging

As shown in Figure 2, our typical mRNA production workflow starts with designing and synthesis of the template DNA sequence with the consideration of preferred codons, GC content, and thermodynamic stability of RNA secondary structures, followed by its cloning into an in vitro transcription vector. The plasmid DNA is then purified, validated, and linearized before being subsequently subjected to the in vitro transcription reaction which results in the generation of the desired transcript. Modified nucleotides, like m1Ψ and m5C, can be incorporated into the in vitro transcription reaction to improve in vivo translation and decrease immunogenicity. Highly efficient capping (>95%) can be achieved either using co-transcriptional or enzymatic approaches. The mRNA is then purified by mRNA-capture beads as the default purification process or oligo dT chromatography upon request. The QC analysis in the table below can be customized for mRNA integrity and purity assessment. Next, mRNA can be further encapsulated in LNP in a microfluidic mixer. The encapsulation efficiency and nanoparticle profiling are analyzed using methods in the table below.

QC Items Methods
mRNA identity Reverse transcription followed by Sanger sequencing
mRNA integrity and purity Denaturing gel electrophoresis, capillary electrophoresis
Capping efficiency LC-MS
PolyA tailing efficiency LC-MS
dsRNA residue ELISA
Template DNA residue PicoGreen staining, qPCR
Protein residue BCA, NanoOrange staining
mRNA encapsulation efficiency RiboGreen staining
LNP particle size, PDI, and surface charge Dynamic light scattering (DLS), TEM
Endotoxin level LAL assay
Sterility Bioburden tests
Experimental validation

The expression and function of our IVT mRNA coding for luciferase and EGFP have been tested in vitro (Figure 3) and in vivo (Figure 4).

Figure 3. Expression of luciferase and EGFP mRNA in 293T or HeLa cells. The mRNA was generated with or without modified nucleotides, N1-Methylpseudouridine (m1Ψ) and 5-Methylcytosine (m5C). Cells grown on the 12-well plates were transfected with 1 ug of mRNA per well. (A) Luciferase activities in 293T cells at 6 h, 24 h, and 48 h post-transfection. Error bars indicate standard deviations. EGFP expression in 293T cells (B) and HeLa cells (C) at 24 h, 48 h, and 72 h post-transfection quantified by flow cytometry. Mean fluorescence intensities are represented by colored bars and percentages of EGFP positive cells are represented by circles, squares, and triangles. Error bars indicate standard deviations. (D) EGFP expression in 293T cells and HeLa cells at 72 h post-transfection observed by microscopy (100X).

Figure 4. Expression of luciferase (Luc) mRNA and mRNA induced immune response in mice. (A) Luciferase activity visualized by live imaging at 6 h,  24 h, and 48 h post-injection. (B) Two pro-inflammatory cytokines, IL-6 and TNF-⍺, were quantified in the serum at 48 h post-injection. Error bars represent standard errors. Mice strain: C57BL/6J; mice age: 8 weeks; injection method: intramuscular injection. (C) IFN-γ ELISpot assay of splenocytes derived from Balb/C mice 14 days post intramuscular injection of 30 ug LNP-encapsulated mRNA coding for viral antigen A, viral antigen B, or control PBS.

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What are the differences between mRNA caps and capping methods?

Cap 0 refers to N7-methylguanosine (m7G) that is added to the 5’ end of eukaryotic mRNAs via a 5’ to 5’ triphosphate linkage. This modification is added via a series of enzymatic reactions that occur co-transcriptionally and functions to regulate nuclear export, transcript stability, and promotes translation of the mRNA through recognition by eukaryotic translation initiation factor (eIF4E). Cap 1 refers to the addition of a methyl group to the 2’O on the first nucleotide (m7GpppNm) of the transcribed mRNA sequence in addition to the m7G cap. In mammalian cells, cap 1 structure is an important marker for mRNA to be recognized as self and not targeted by innate immunity. Adding cap 1 structure to synthesized mRNA has been demonstrated to enhance mRNA expression in vivo and reduced its immunogenicity.

Capping for in vitro transcribed RNA can occur either co-transcriptionally with cap analogs or post-transcriptionally via enzymatic reactions. We offer both capping methods, and the efficiency of them has been well validated using LC-MS. Depending on the client-preferred capping method, we will choose a compatible backbone for cloning the IVT mRNA vector from the beginning.

Why should I consider incorporating modified nucleotides in mRNA and which ones can be included?

Cells contain cytosolic and endosomal RNA receptors that activate the immune response upon recognition of foreign RNA. Modified nucleotides are commonly found in endogenous cellular RNA. Incorporating certain modified nucleotides in IVT mRNA reduces its immunogenicity, alters secondary structure, and increases translation efficiency and half-life in a sequence-dependent manner. We provide a wide range of modified nucleotides, including the commonly used N1-Methylpseudouridine (m1Ψ) and 5-Methylcytosine (m5C). N1-Methylpsuedouridine and 5-Methylcytosine are naturally occurring nucleotides that were first identified in tRNAs, however, their use in coding mRNAs has only recently been appreciated. These methylated derivatives of uridine and cytosine can replace their canonical nucleotides in mRNA IVT and translation without altering traditional Watson-Crick base pairing. A major advantage to their use in mRNA therapeutics is their ability to alter recognition by RNA immune receptors thus mitigating unwanted immune effects and enhancing transcript stability and translation.