Messenger RNA (mRNA) is a crucial intermediary molecule in biological systems. It carries genetic instructions copied from DNA, acting as a temporary blueprint for building proteins within cells. Proteins perform a vast array of functions, from structural support to enzymatic reactions. Manufacturing specific mRNA molecules outside of living cells has opened new avenues in medicine and research, allowing scientists to direct cells to produce desired proteins. This process involves several precise steps to ensure the resulting mRNA is suitable for its intended purpose.
DNA Template Creation
mRNA manufacturing begins with creating a specific DNA template. This template contains the genetic sequence to be copied into mRNA, along with necessary regulatory elements. It can be a circular plasmid, grown in bacteria for large quantities, or linear DNA fragments generated by polymerase chain reaction (PCR).
The DNA template is designed to include the gene of interest, which encodes the protein, and a promoter region. This promoter acts as a starting signal for the enzyme that synthesizes the mRNA. A sequence for a poly-A tail, which contributes to mRNA stability, is also incorporated. The accuracy and quality of this DNA blueprint are fundamental, as they dictate the characteristics of the final mRNA product.
In Vitro Transcription
With the DNA template prepared, the next step is in vitro transcription. This process synthesizes mRNA in a controlled laboratory environment, mimicking natural transcription. Key ingredients include the DNA template, specific RNA polymerase enzymes, and nucleotide building blocks.
RNA polymerase recognizes the promoter sequence on the DNA template. It unwinds a segment of the DNA double helix and moves along one strand, reading its sequence. As it progresses, the enzyme adds complementary ribonucleotides to form a new, single-stranded mRNA molecule. This results in a direct copy of the genetic information from the DNA template into RNA.
mRNA Processing and Purification
After synthesis, mRNA undergoes modifications to become functional and stable. Two modifications are the addition of a 5′ cap and a poly-A tail. The 5′ cap, a modified guanosine nucleotide, is added to the beginning of the mRNA strand. This cap protects the mRNA from degradation and aids its recognition by cellular machinery for protein synthesis.
A poly-A tail, consisting of many adenine nucleotides, is added to the 3′ end. This tail also protects the mRNA from breakdown and plays a role in its transport and efficient translation into protein. Following these modifications, the mRNA is purified to remove impurities. Methods like chromatography or tangential flow filtration separate the mRNA from residual DNA template, enzymes, and truncated RNA strands. This purification ensures a pure and effective mRNA product.
Lipid Nanoparticle Encapsulation
Once processed and purified, mRNA often requires protection and a delivery system, especially for therapeutic applications. Unprotected mRNA is fragile and easily degraded by enzymes in the body. Lipid nanoparticles (LNPs) are commonly used as protective vehicles. They encapsulate the mRNA, shielding it from degradation and facilitating its entry into target cells.
LNPs are composed of four main types of lipids: ionizable lipids, helper phospholipids, cholesterol, and PEGylated lipids. Ionizable lipids are designed to be positively charged in acidic environments, allowing them to bind to the negatively charged mRNA. These components self-assemble around the mRNA, forming a stable nanoparticle structure that efficiently delivers its genetic cargo into cells.
Quality Control and Storage
The final stages of mRNA manufacturing involve quality control (QC) and proper storage. QC measures are performed throughout the process and on the finished product to ensure safety and effectiveness. Tests verify mRNA purity, checking for contaminants like residual DNA, proteins, or unwanted RNA fragments. The integrity of the mRNA, confirming it is full-length and not degraded, is also assessed.
Other tests determine the mRNA concentration and ensure the product is sterile. Proper storage conditions are important to maintain mRNA stability over time. mRNA products, especially those encapsulated in LNPs, often require ultra-cold temperatures, sometimes as low as -70°C or below, to prevent degradation and preserve their activity. These conditions are necessary to ensure the product maintains its quality until use.