Nature Biotechnology: Engineered RNA polymerases to purify mRNA production and reduce immunostimulation byproducts

mRNA therapy is an emerging drug with some advantages
over large molecule biologics and small molecule drugs.
From a manufacturing perspective, a key advantage of mRNA therapeutics is the use of a single, standardized manufacturing process for a range of targets
.

To ensure high purity of mRNA during therapeutic applications for repeated or long-term administration, several costly and time-consuming purification steps
are required.
These additional steps often result in increased RNA degradation, quality degradation, and/or yield degradation, so a more efficient mRNA synthesis process is needed to reduce the burden
of downstream purification steps.

On November 10, 2022, researchers from Moderna published a research paper in the journal Nature Biotechnology entitled: An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts
。

The research team developed a dual mutant of T7 RNA polymerase through rational design, which can simplify the mRNA production process of in vitro transcription (IVT) compared to wild-type T7 RNA polymerase, improve the purity of mRNA products without reducing yield, significantly reduce the production of immunostimulatory byproduct double-stranded RNA (dsRNA), and also have faster production time
.

This discovery opens the door
to streamlining the development of therapeutic mRNA production processes, improving cost-effectiveness, and producing mRNA-based products with clinical or commercial applications.

In vitro transcription (IVT) is an enzymatic process
used to synthesize mRNA products.
During the IVT process, RNA polymerase (RNAP) is responsible for transcribing RNA
from the DNA template.
Although IVT is an accepted process in research laboratories, optimization for industrial-scale production has only recently begun
.

The most commonly used polymerase for in vitro transcription (IVT) is T7 RNA polymerase with a molecular weight of about 99 kDa
.
Due to its high yield and high fidelity of transcripts, it is well suited for in vitro RNA production, so it has been widely used
in scientific research and commercial development.

However, T7RNA polymerase also produces multiple byproducts, including immunostimulating byproducts double-stranded RNAs (dsRNAs) produced from template transcription, which may affect efficacy and safety, especially in therapeutic applications
.

The production of double-stranded RNA (dsRNAs) and other by-products during in vitro transcription (IVT) is influenced
by the T7 RNA polymerase conformation in the catalytic cycle.
T7 RNA polymerase-mediated transcription consists of three distinct phases: initiation, extension, and termination
.
During revelation, the N-terminal domain (NTD) of the T7 RNA polymerase binds to the promoter sequence to form a initiation complex (IC).

This starting complex is unstable and produces short RNA transcripts (2-10 nucleotides in length).

When transcript lengths greater than ~10 nucleotides, promoter binding residues in NTD rearrange, releasing DNA promoter regions to form a stable, procedural enzyme extension complex (EC).

Termination, on the other hand, occurs in response to a specific signal sequence or when it reaches the end of a linearized DNA template, a process known as "runaway transcription" that typically produces full-length RNA, but sometimes produces non-template appendages
at its 3' end.

In addition, T7 RNA polymerase has the ability to transcribe RNA-templated products, resulting in double-stranded RNA (dsRNAs) products and looped dsRNA products
.

The exact mechanism behind this 3' end heterogeneity and looping dsRNA formation is unknown
.
Therefore, it is challenging
to design a sound way to prevent both behaviors.

Double-stranded RNA (dsRNAs) molecules are innate immune response activators that can be recognized by RIG-I, MDA5 and LGP2 to trigger the body's innate immune response
.
Therefore, these dsRNA molecules affect the potency and safety of the mRNA product, requiring additional purification steps to remove the dsRNA from the final mRNA
product.

Two methods are commonly used to reduce dsRNA in mRNA products during production, one is to purify mRNA products by chromatography, such as reversed-phase high performance liquid chromatography (RP-HPLC) or cellulose-based dsRNA isolation; The second is to optimize in vitro transcription (IVT) conditions to reduce by-product formation
.

Modifying in vitro transcription (IVT) conditions can effectively reduce the dsRNA burden, but is not sufficient to eliminate the need for downstream purification of mRNA products by reversed-phase high performance liquid chromatography (RP-HPLC), an expensive and time-consuming step
.

In this study, the research team reported a double-mutant T7 RNA polymerase (G47A+884G), which can significantly reduce dsRNA byproducts in in vitro transcription (IVT) while maintaining mRNA yield and purity, thereby reducing the burden
of controlling transcription-related dsRNA byproducts during downstream purification.

Overall, this study shows that the formation
of immunostimulatory byproducts can be reduced by modifying the T7 RNA polymerase.
This discovery opens the door
to streamlining the development of therapeutic mRNA production processes, improving cost-effectiveness, and producing mRNA-based products with clinical or commercial applications.

The paper was co-authored by Amy Rabideau of Moderna, and the first authors were Athanasios Dousis (currently joined Tessera) and Kanchana Ravichandran
.

Original source:

Dousis， A.
， Ravichandran， K.
， Hobert， E.
M.
et al.
An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts.
Nat Biotechnol (2022).
https：//doi.
org/10.
1038/s41587-022-01525-6.