Moderna, Inc., Cambridge, MA, USA.
Tessera Therapeutics, Somerville, MA, USA.
Nat Biotechnol. 2023 Apr;41(4):560-568. doi: 10.1038/s41587-022-01525-6. Epub 2022 Nov 10.
In vitro transcription (IVT) is a DNA-templated process for synthesizing long RNA transcripts, including messenger RNA (mRNA). For many research and commercial applications, IVT of mRNA is typically performed using bacteriophage T7 RNA polymerase (T7 RNAP) owing to its ability to produce full-length RNA transcripts with high fidelity; however, T7 RNAP can also produce immunostimulatory byproducts such as double-stranded RNA that can affect protein expression. Such byproducts require complex purification processes, using methods such as reversed-phase high-performance liquid chromatography, to yield safe and effective mRNA-based medicines. To minimize the need for downstream purification processes, we rationally and computationally engineered a double mutant of T7 RNAP that produces substantially less immunostimulatory RNA during IVT compared with wild-type T7 RNAP. The resulting mutant allows for a simplified production process with similar mRNA potency, lower immunostimulatory content and quicker manufacturing time compared with wild-type T7 RNAP. Herein, we describe the computational design and development of this improved T7 RNAP variant.
体外转录 (IVT) 是一种基于 DNA 的长 RNA 转录物合成过程，包括信使 RNA (mRNA)。对于许多研究和商业应用，由于噬菌体 T7 RNA 聚合酶 (T7 RNAP) 能够高保真地产生全长 RNA 转录物，因此通常使用 T7 RNAP 进行 mRNA 的 IVT；然而，T7 RNAP 也可以产生双链 RNA 等免疫刺激性副产物，这些副产物可能会影响蛋白质表达。这些副产物需要使用反相高效液相色谱等方法进行复杂的纯化过程，才能得到安全有效的基于 mRNA 的药物。为了尽量减少下游纯化过程的需要，我们通过合理和计算的方式对 T7 RNAP 进行了双突变，使其在 IVT 过程中产生的免疫刺激性 RNA 明显少于野生型 T7 RNAP。与野生型 T7 RNAP 相比，产生的突变体具有类似的 mRNA 效力、更低的免疫刺激性含量和更快的制造时间，因此简化了生产过程。本文描述了这种改进的 T7 RNAP 变体的计算设计和开发。
