1. This study examined how lipid nanoparticle (LNP) formulation and mRNA modifications impact delivery efficiency in normal versus Niemann-Pick C1 (NPC1)-deficient cells. 2. The results showed that an optimized LNP for mRNA delivery was less effective in NPC1-deficient cells, while the original LNP used for siRNA had higher efficacy in NPC1-deficient cells. This suggests the formulations impact trafficking pathways. 3. Modifying LNP composition, such as reducing PEG percentage or changing phospholipid, reversed the efficacy pattern by formulation. mRNA modifications also impacted trafficking.

1. 1Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge MA, 2Koch Institute
for Integrative Cancer Research, Cambridge MA, 3Department Of Pharmaceutical Sciences, College of
Pharmacy, Oregon State Univ. Portland OR
Niemann-Pick C1 impact on mRNA-containing lipid nanoparticle endocytosis
Anastasia N. Neuman1,2, James C. Kaczmarek1,2, Gaurav Sahay3
Daniel G. Anderson1,2
Lipid Nucleic Acid Delivery
Numerous diseases, including cancer, heart disease, diabetes, and the
common cold, 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; however the
cellular processes that determine their effectiveness remain unclear2.
mRNA Delivery with LNPs
REFERENCES
Approach
This work was supported by Shire
Pharmaceuticals, the MIT
Koch Institute for Integrative Cancer
Research, and the C. Michael Mohr
Scholarship for Undergraduate
Research.
siRNA Delivery with LNPs3
mRNA Modification Impact on Efficacy
Conclusions
Potential for Future Studies
ACKNOWLEDGMENTS
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 siRNA, 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, with the original particle likely endocytosed through
macropinocytosis and the optimized particle using an NPC1-dependent
pathway.
Formulation Modification Impact on Efficacy
Changing the PEG percentage from 2.5% to 1.5% results in the particles
being most effective in NPC1-/- cells, as observed with the original
formulation. PEG reduces LNP aggregation and prevents nonspecific
endocytosis by immune cells1. Reducing the PEG percentage may allow the
particle to use multiple endocytotic pathways, and reduce reliance on NPC1
for cell entry.
Changing the phospholipid from DOPE to DSPC also reversed the pattern.
DOPE has a primary amine headgroup and and a tail with one degree of
unsaturation while DSPC has a quaternary amine headgroup and fully
saturated tail2. Conical phospholipids like DOPE adopt a hexagonal phase
with lower stability while cylindrical phospholipids like DSPC adopt a more
stable lamellar phase2. The unstable hexagonal phase of DOPE may
promote endosomal escape. The more stable lamellar phase of DSPC
inhibits endosomal escape and would depend on NPC1 deficiency to allow
more LNPs to accumulate in the endosomes.
Unlike the luciferase
mRNA provided by Shire
Pharmaceuticals used in
the prior experiments,
unmodified luciferase
mRNA, luciferase mRNA
5-methylcytosine (5mc)
and luciferase mRNA
purchased from Tri-Link
with both and 5-
methylcytosine and
psuedouridine (5mc psu)
show higher efficacy in
NPC1- deficient cells than
normal cells in both the
original and the optimized
LNP. This could be due to
differences in mRNA
folding driven by these
modifications.
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.
In conclusion, our work has demonstrated the following:
• The PEG percentage and phospholipid of mRNA-loaded LNPs may
impact the trafficking method.
• Modifications in mRNA may impact trafficking of mRNA-loaded
particles.
• Use imaging to observe how changes in LNP formulation and mRNA
modifications affect the trafficking of particles.
• Optimize an mRNA-loaded LNP for treatment of Neimann Pick Type I
Disease, the disease after which NPC1 is named.
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 two main goals of this
project are as follows:
1) Observe the effect of LNP formulation differences on mRNA trafficking
and delivery
2) Observe the effect of mRNA modifications on trafficking and delivery
These experiments were conducted using luciferase mRNA. 24 hours
following in vitro transfection of wild-type and NPC1 knockout mouse
embryonic fibroblast (MEF) cells (100 ng mRNA/well), the cells were lysed
and luciferase expression was quantified using luminescence.
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, most particles are recycled through either transport to the
ER-Golgi route or direct fusion by the endosome to the plasma
membrane3. In NPC1-deficient cells the 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
Previous work has shown that siRNA-loaded lipid nanoparticles are
recycled back out of a cell following endocytosis, rendering a large
proportion of the delivered dose ineffective3. This recycling is dependent
on the presence of Niemann-Pick type C1 (NPC1), a protein involved in
cellular cholesterol trafficking4. Without NPC1, the recycling pathways of
the late endosomes are largely inhibited. Here, we see that AF647
siRNA delivery and gene silencing in NPC1-deficient cells is increased
over that in wild-type cells3.
These siRNA experiments were done using a C12-200 based lipid
formulation, which is here referred to as the “original particle”.
Experimental design has resulted in a particle optimized for mRNA
delivery, here referred to as the “optimized particle” (Table 1). The
optimized particle was found to perform no better for delivery of siRNA2,
and the physical basis for the difference in efficacy is unknown.
cationic lipid
phospholipid
polyethylene glycol (PEG)
cholesterol
Fig. 3: Gene silencing in wild-type and NPC1
deficient cells3.
Uridine Pseudouridine
e
Cytidine 5-methylcytidine
Fig. 7: mRNA base modifications.
i) ii)
Fig. 4: luciferase mRNA delivery by LNPs in wild-type
and NPC1 deficient cells.
Fig. 5: Impact of formulation changes on luciferase
mRNA delivery in wild-type and NPC1-deficient cells.
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.
Fig. 6: mRNA modification impact on luciferase mRNA
delivery in wild-type and NPC1-deficient cells.