Poster from Amgen program summer 2017.

1. Conclusions
1Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge MA, 2Koch Institute for Integrative Cancer
Research, Cambridge MA
mRNA-containing lipid nanoparticle formulation impact on endocytosis
Anastasia N. Neuman1,2, James C. Kaczmarek1,2, Daniel G. Anderson1,2
Lipid Nucleic Acid Delivery
Numerous diseases, including cancer, heart disease, and diabetes,
are brought on by aberrant protein expression. Delivery of nucleic
acids to control such aberrant protein expression represents a
promising therapeutic strategy to treat disease at its source, but
nucleic acids typically require the use of delivery vehicles to be useful
in a therapeutic context. Lipid nanoparticles (LNPs), in particular, have
been shown to effectively deliver nucleic acids such as siRNA and
mRNA both in vivo and in vitro1. Highly potent materials have been
developed through the use of high throughput screening techniques;
one such example of this for mRNA delivery is the optimized
formulation found in Table 12. However, the cellular processes that
determine their effectiveness remain unclear2. In particular, what
makes the optimized formulation more effective for mRNA delivery
than the original formulation in Table 1, which has previously been
used for siRNA delivery3, is unknown.
mRNA Delivery with LNPs
Approach
This work was supported by RaNA
Therapeutics, the MIT Koch Institute for
Integrative Cancer Research, and the Amgen
Foundation.
Formulation Impact on Endocytosis
Fig. 1: Nucleic acids encapsulated by a stable lipid
nanoparticle (LNP) formulated with a cationic lipid,
phospholipid, PEG, and cholesterol1.
NPC1 Facilitated Endocytic Recycling
When delivered with an LNP optimized for mRNA delivery, a
lower efficacy was observed in NPC1-deficient cells than
normal cells. Upon delivery with the original LNP used in the
study involving siRNA3, the original pattern was observed,
with higher efficacy in NPC1-deficient cells than normal cells.
This pattern suggests that the formulation changes impact
the subcellular trafficking of the LNPs.
To further investigate the impact LNP formulation has on
endocytosis, LNPs were delivered to cells treated with an
inhibitors of endocytotic pathways and the efficacy was observed.
U18666A, a drug known to inhibit cholesterol trafficking5, is
shown to decrease efficacy only in the optimized particle.
Efficacy significantly increases in both the original and original
PEG % formulations, suggesting that the change in PEG % may
cause the optimized particle’s reliance on the cholesterol
trafficking pathway.
1. K. J. Kauffman, M. J. Webber, D. G.
Anderson, Journal of Controlled Release,
2016, 240, 227-234.
2. K. J. Kauffman, J. R. Dorkin, J. H. Yang,
M. W. Heartlein, F. DeRosa, F. F. Mir, O.
S. Fenton, D. G. Anderson, Nano Lett.
2015, 15, 7300–7306.
3. G. Sahay, W. Querbes, C. Alabi, A.
Eltoukhy, S. Sarkar, C. Zurenko, E.
Karagiannis, K. Love, D. Chen, R. Zoncu,
Y. Buganim, A. Schroeder, R. Langer, D.
G. Anderson, Nature Biotechnology,
2013, 31, 653-658.
4. E. Lloyd-Evans, A. J. Morgan, X. He, D.
A. Smith, E. Elliot-Smith, D. J. Sillence,
G. C. Churchill, E. H. Schuchman, A.
Galione, F. M. Platt, Nature Medicine,
2008, 14, 1247-1255.
5. A. Eltoukhy, G. Sahay, J. M.
Cunningham, D. G. Anderson, ACS
Nano, 2014, 8, 7905-7913.
The change in PEG % and
change in phospholipid may
be the cause of the efficacy
differences between the
original and optimized LNPs.
In particular, the change in
PEG % may be involved in
the optimized LNPs apparent
dependency on NPC1.
Future studies will involve
FACS analysis to determine
the effect of these inhibitors
on LNP uptake into cells and
investigation into the cause
of efficacy differences seen
with inhibitors other than
U18666A.
We hypothesize that differences in subcellular trafficking may
account for some of the observed differences in efficacy
between different nanoparticle formulations and nucleic acid
cargos. As such, the main goal of this work is to observe the
effect of LNP formulation differences on mRNA trafficking
and delivery.
For experiments involving endocytosis inhibitors cells were
pretreated with inhibitors (5 𝜇M chlorpromazine, 50 𝜇M
dynasore, 10 𝜇M EIPA, 5 𝜇M filipin, 10 𝜇M genistein, and 5 𝜇
M U18666A) 1 hour prior to transfection.
Fig. 2: LNP trafficking in i) NPC1+/+ cells and ii) NPC1-/- cells3.
One cellular process known to impact the effectiveness of siRNA-
loaded LNPs is endocytic recycling. siRNA-loaded LNPs often enter
cells through macropinocytosis and the majority are directed to late
endosomes. The siRNA must escape these endosomes for gene
silencing to occur. In normal cells, particles are recycled through
transport to the ER-Golgi route or endosomal fusion to the plasma
membrane3. In NPC1-deficient cells LNPs are not recycled and
accumulate in the late endosomes, allowing siRNA to continuously
escape3. Thus, NPC1-deficient cells show increased gene silencing of
the target gene3.
Original
formulation
Optimized
formulation
C12:200:mRNA
weight ratio
5:1 10:1
phospholipid DSPC DOPE
C12-200 molar
composition
50% 35%
phospholipid
molar
composition
10% 16%
cholesterol molar
composition
38.5% 46.5%
C14 PEG 2000
molar
composition
1.5% 2.5%
Table 1: Original and Optimized LNP Formulations2
cationic lipid
phospholipid
polyethylene glycol (PEG)
cholesterol
i) ii)
Fig. 3: luciferase mRNA delivery by
LNPs with two different formulations in
wild-type and NPC1 deficient cells
(mean ± SD, n=4); *** indicates p < .001.
Fig. 4: Impact of formulation changes
on luciferase mRNA delivery in wild-
type and NPC1-deficient cells (mean ±
SD, n=4); * indicates p < 0.05, **
indicates p < .01, *** indicates p < .001.
Fig. 5:Endocytosis inhibitor impact on transfection efficiency of four different LNP
formulations (mean ± SD, n=4); * indicates p < 0.05, ** indicates p < .01, *** indicates p < .001,
compared to no inhibitor control.
The formulation of the LNPs was
modified by one factor at a time
from the optimized formulation to
the original formulation. The
optimized and original formulations
can be found in Table 12. Changing
the PEG percentage from 2.5% to
1.5% and changing the
phospholipid from DOPE to DSPC
results in the particles being most
effective in NPC1-/- cells, as
observed with the original
formulation.
ACKNOWLEDGEMENTS
REFERENCES
In vitro transfectionLNP formulation Luciferase assay