The invisible battle behind RNA delivery

A new study using advanced microscopy techniques has revealed why lipid nanoparticles (LNPs), used to deliver RNA in vaccines and gene therapies, often fail to deliver their cargo effectively inside cells. Researchers discovered that lipids and RNA often separate inside cells, and the cell's repair system actively hinders RNA escape, limiting therapeutic efficacy. Understanding these barriers is crucial for designing more effective RNA delivery systems.

Read the full article at https://www.paperleap.com/blog/articles/the-invisible-battle-behind-rna-delivery-0cccua

Episode Transcript

Speaker 1 (00:00):
Welcome to the paper Leap podcast, where science takes the mic.
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(00:22):
to subscribe so you never miss an insight. All the
content is also available on paperleap dot com. Okay, ready,
let's start. If you've had an mRNA vaccine, you've already
experienced one of the most impressive accomplishments in modern medicine.
Scientists packaging fragile genetic instructions inside tiny lipid bubbles called

(00:45):
lipid nanoparticles or LNPs to deliver them safely into yourselves.
But while these microscopic capsules have transformed how we fight viruses,
they still face a frustrating problem. Most of the RNA
they carry never reaches its destination inside the cell. A
new study published in Nature Communications by Johanna Maria Johansson, Hampis,

(01:10):
du Riets, Hampis, Headlund, Hannah Ericsson, and Anders Vitrup at
Lund University in collaboration with Astrazenica's Biopharmaceuticals R and D
teams in Sweden and the US dives deep into this mystery.
Using a combination of live cell and super resolution microscopy.

(01:31):
The team watched in real time how RNA loaded nanoparticles
behave once they inter cells and uncovered a series of
invisible roadblocks that explain why these vehicles are so inefficient
at delivering their genetic cargo. Lipid nanoparticles have been hailed
as molecular couriers. They carry snippets of genetic code, either

(01:53):
mRNA used in vaccines and gene therapy, or sRNA used
to silence disease causing genes, wrapped in a fatty shell.
Once injected into the bloodstream, they traveled to target cells,
fuse with their outer membrane, and ideally release the RNA
into the cell's interior the sitozol, where it can do

(02:15):
its job but ideally is doing a lot of heavy lifting.
While l andps work well in the liver, where they
were first approved for treating rare genetic diseases, they struggle
in most other tissues. The RNA gets trapped inside bubble
like compartments called endosomes, internal mail rooms, where the cells
sorts and digests incoming material. For the RNA to work,

(02:38):
it needs to escape these compartments into the sitozol, something
that rarely happens until now. Scientists knew this escape step
was the main bottleneck. What they didn't know was why
it's so inefficient. That's where Johansson and colleagues stepped in.
Using state of the art microscopes, the LUN team labeled

(02:58):
both the RNA and the lipid shell of LNPs with
fluorescent dyes. This allowed them to literally watch the nanoparticles
move through cells, merge with endosomes, and sometimes, but not often,
burst free. One of their key tools was a molecule
called galectin nine, a kind of damage detector that glows

(03:19):
when an endosome's membrane is breached. When LNPs disrupted an endosome,
galectin nine would light up, revealing a moment of membrane damage,
potentially a portal for RNA escape. But the researchers quickly
realized something surprising. Not all damaged endosomes actually released RNA.

(03:40):
In fact, many ruptured endosomes contained no detectable RNA at all.
It seemed that even when the nanoparticles broke through cellular barriers,
their genetic cargo often failed to come along for the ride.
By tracking both components of the LNP, the ionizable lipid
which helps the particle fel use with membranes, and the

(04:01):
RNA payload, the team discovered that these two ingredients often
go their separate ways once inside the cell. As the
endozome matures, the nanoparticle starts to fall apart, and the
lipids and RNA drift into different subcompartments. In some cases,
the lipid accumulates at the endosomal membrane, causing damage or leaks,

(04:23):
while the RNA remains trapped elsewhere. It's as if the
delivery truck crashes through the warehouse wall, but the package
stays inside the box. This explains why the researchers saw
so many damaged endosomes that were strangely empty. The lipid
could still harm the membrane even after its RNA payload
had wandered off. Cells don't take membrane damage lightly. They

(04:46):
have an inbuilt repair system known as the s cart machinery,
that rushes into patch holes in seal leaks before too
much escapes. The researcher's team found that this repair system
kicks in quickly when LNP disturbed the endosomal membrane, sometimes
so quickly that it stops the RNA from escaping altogether.

(05:07):
When the researchers silenced key s cart genes, more damage
signals appeared in the cells, suggesting that the repair machinery
was indeed limiting RNA delivery. The cell, in essence, was
fighting back against the therapeutic invasion. Even when everything went right,
the l and P entered the cell, reached the right compartment,

(05:28):
caused a rupture, and avoided rapid repair, only a small
fraction of the RNA actually escaped into the cytosol. The
process was fast, often happening within seconds, but incomplete. Many
nanoparticles remained tethered to the endosomal membrane, leaking out only
a portion of their payload before being sealed off or digested.

(05:51):
What's more, larger mRNA molecules fared worse than smaller sRNA ones.
While up to half of sRNA molecules could escape from
damage endosomes, fewer than one in five mRNA containing endosomes
released detectable cargo. The bigger, bulkier strands simply had a
harder time squeezing through the same tiny holes. The team's

(06:14):
microscopic images are almost cinematic. They show glowing RNA specs
clustering near membranes, flickering as the endozome wall ruptures, and
then fading as some RNA drifts into the cell's interior.
In other frames, bright lipid patches appear like fireflies on
the membrane's edge, areas where the ionizable lipid has accumulated

(06:36):
and caused stress. At times, the lipid and RNA appear
side by side, almost touching. In others they drift apart completely.
These visual cues reveal a chaotic dance between particle disintegration,
membrane damage, and cellular repair, a dance that determines whether
the therapy works or fails. Are the workhourses of today's

(07:01):
RNA therapeutics. They made mRNA vaccines possible, but scientists want
to go much further, using l and ps to deliver
RNA that repairs genes, kills cancer cells, or regenerate damage tissues.
To reach those goals, l andps must reliably deliver their
cargo into many different cell types, not just liver cells.

(07:24):
By pinpointing exactly where and why RNA delivery fails. Joansen
and colleagues have provided a roadmap for improvement. Their findings
suggest that to make L andps more effective, scientists need
to prevent lipid RNA separation inside endosomes so both parts
stay together until release, control the timing and extent of

(07:46):
membrane damage, balancing escape with cell safety, and temporarily modulate
the cell's repair system, giving RNA a better chance to
get out before the s cart machinery patches the whole.
In other words, future generations of L and p's will
need to be smarter couriers, able to sense when to

(08:06):
open the package and where to deliver it without triggering
the cell's emergency response. The approach of the team, which
combines live cell imaging with biophysical analysis, could soon be
used to test new types of nanoparticles, including those made
with different lipids like ALC thirteen fifteen used in Pfizer's
COVID nineteen vaccine, or SM ten used by Maderna. These

(08:30):
variants were engineered to improve RNA delivery, but no one
has yet seen exactly how they perform inside living cells
at this level of detail. Ultimately, this line of research
could usher in a new era of rational design for
RNA delivery systems, moving away from trial and error chemistry
towards evidence based engineering guided by what actually happens inside

(08:55):
the cell. What Joannsen and her colleagues have shown is
that inside NA every cell, a microscopic battle plays out
between the nanoparticle, its fragile RNA cargo, and the cell's
own defenses. Most of the time the cell wins, but
thanks to this research, we now know what needs to
change for the RNA to stand a fighting chance. That's

(09:20):
it for this episode of the paper Leap podcast. If
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