APR Sept/Oct 2023 - 95

« INTERVIEW
The ARCA method is one of the original capping approaches. This
co-transcriptional method creates a cap 0 (non-self) structure with
approximately 80 percent capping efficiency. On the other hand,
enzymatic capping involves post-transcriptional capping using
capping enzymes to produce a cap 0 or cap 1 structure, depending on
the enzymes used. While this method requires multiple reactions and
purification steps, these can be optimized for more straightforward
processing. Finally, CleanCap® technology is a proprietary cap analog
producing a cap 1 (self) structure incorporated by RNA polymerase
in a single in vitro transcription reaction. Additionally, it is available
in several various cap structures, include CleanCap®
AG 3' OMe
and CleanCap® M6 for improving protein translation, along with
CleanCap® AU specifically for saRNA. The design of this method offers
several advantages by increasing capping efficiency while reducing
process time.
3. How does mRNA capping ensure the
stability and efficiency of mRNA molecules in
vaccine and therapeutic development?
Natural mRNA caps made endogenously within the cell have a primary
function of shuttling immature RNA out of the nucleus and facilitating
mRNA processing and maturation. However, when mRNA has been
produced exogenously, such as in the context of mRNA-based vaccine
and therapeutics development, the cap has another critical role. In
this case, capping affords therapeutic mRNA molecules a high degree
of stability by preventing its rapid degradation once inside the cell,
enabling the mRNA to maintain its therapeutic function and potency.
Furthermore, caps with a cap 1 structure do not trigger an immune
reaction and therefore do not detract from the efficacy of the vaccine
or therapeutic product.
Additionally, regarding manufacturing, different capping methods
can also improve how efficiently manufacturers can produce a
vaccine or therapeutic product. For example, CleanCap® technology's
one-pot solution reduces manufacturing steps allowing for quicker
turnaround times, easier scale-up, and higher transcriptional yields
than other methods - all which have an overall positive impact on
manufacturing costs.
4. What are the advantages and limitations
of different mRNA capping technologies for
producing vaccines and therapeutics?
Each method of mRNA capping offers different advantages and
disadvantages. For example, the ARCA approach tends to provide
lower transcriptional efficiency and produces a cap 0 structure that
is not recognized as self in eukaryotes and thus can cause higher
immunogenicity. On the other hand, enzymatic capping can achieve
higher capping efficiency rates than ARCA, but it usually requires
additional purification steps. In contrast, CleanCap® technology
produces a natural cap 1 structure,
reducing immunogenicity.
Moreover, it is a one-pot solution with fewer manufacturing steps that
caps RNA molecules at about 95 percent efficiency.
The importance of choosing the correct mRNA capping technology
extends beyond just having technical implications. Manufacturers
should remember that a capping reagent impacts both downstream
and upstream processes, impacting cost, time, yield, and purity- all of
which affect a vaccine or therapeutic's journey to market.
5. How do mRNA capping technologies impact
the immunogenicity and efficacy of mRNAbased
vaccines and therapeutics?
A vaccine is a molecule which stimulates the body to mount an immune
response against a pathogen of entry, whether a bacterium or a virus.
Critical to effective vaccine design is ensuring that the body's immune
system only responds to the specific gene sequence responsible for
a particular disease but not other vaccine components. As such, all
capping components must be immune-silent to ensure the body
generates the desired immune response, limiting the potential for offtarget
effects and immunogenicity. Since an uncapped 5'-phosphate
mRNA is immunogenic, it is critical that the mRNA is capped so it does
not cause immune response. The cap binds to the mRNA translation
initiation factor and initiates the mRNA translation.
In practice, vaccine development requires delicately balancing potency
with specificity. The goal is to generate an immune response specific to
the pathogen of entry and not the vaccine itself.
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