Lipid nanoparticle delivery of RNA for loss-of-function and

Lipid nanoparticle delivery of RNA for loss-of-function and gain-offunction studies in primary neurons in vitro and in vivo.
David Zwaenepoel1, A. S. Ansari1,2, A. Thomas1, C. Walsh1, T. Leaver1, A. Wild1,2, K. Ou1,2, J. Taylor1, E. Ramsay1,2
1Precision
NanoSystems, Inc., Vancouver, Canada.
Summary
2University of
British Columbia, Vancouver, Canada
in vitro
SUB9KITS™ RNA-Lipid Nanoparticles:
Exploit low-density lipoprotein (LDL) receptormediated uptake to transfect primary neurons with
high efficiency (> 95%)
SUB9
SUB9 KITS™ siRNAsiRNA-Lipid Nanoparticles Mediate >90%
90% Target Gene
Knockdown in Primary Rat Cortical Neuron Cultures.
Cultures.
A high efficiency, low toxicity transfection modality
loss-of-function and gain-of-function studies in
primary neurons in vitro and in vivo
SUB9
siRNA-Lipid Nanoparticles Maintains >70%
SUB9 KITS™ siRNA
70% Target Gene
Knockdown in Primary Rat Cortical Neuron Cultures for 21 Days.
Days.
Mediate prolonged (>21 days), dose-dependent target
gene knockdown in primary neurons
Enable simultaneous knockdown of multiple gene
targets in primary neurons
Manufacture & Mechanism-of-Action
Pten Expression
(% Control)
Promote mRNA-mediated protein expression in primary
neurons
Primary E18 rat cortical neurons (DIV 13; mixed
culture) were treated with SUB9KITS™ siRNALipid Nanoparticles (10 ng/mL & 100 ng/mL
siRNA targeted against Pten gene, or 100
ng/mL non-targeted siRNA control). 72 h posttreatment, Pten mRNA (RT-qPCR) and PTEN
protein (Western Blot) were quantified. Cell
viability,
as
determined
by
lactose
dehydrogenase assays, indicated no difference
between treated and control cells (data not
shown).
SUB9
SUB9 KITS™ siRNAsiRNA-Lipid Nanoparticles Mediate Signi fica nt
Reduction in Target Protein Knockdown up to 1 mm from
the Injection Site Following Direct Injection into Rat
Somatosensory Cortex.
Cortex.
Primary E18 rat cortical neurons (DIV 13; mixed
culture)
were
treated
with
a
single
administration of SUB9KITS™ siRNA-Lipid
Nanoparticles (100 ng/mL siRNA targeted
against Hprt gene, or 100 ng/mL non-targeted
siRNA control). Hprt mRNA (RT-qPCR) was
quantified of Days 1, 3, 6, 10, 14 and 21 posttreatment.
SUB9
SUB9 KITS™ RNARNA-Lipid Nanoparticles are Manufactured
Using Microfluidics
in vivo
Lipid mix
Sprague-Dawley rats received a single injection of SUB9KITS™ siRNA nanoparticles
(500 nl at 5 mg/mL siRNA for 10 minutes) directly into the somatosensory cortex.
Cortical slices were prepared 5 days post-injection, and were analyzed by Western
Blot. The results showed a significant reduction in target PTEN protein (> 80%).
Conclusion
SUB9
SUB9 KITS™ siRNAsiRNA-Lipid Nanoparticles Mediate Simultaneous Multiple
Target Gene Knockdown in Primary Rat Cortical Neuron Cultures.
Cultures.
RNA
150
SUB9KITS™ RNA-Lipid Nanoparticles are manufactured using customized
microfluidic mixing devices. Microfluidic mixing mediates production of welldefined neutral RNA-Lipid Nanoparticles that posess a “solid-core” morphology.
in vitro
Paracrine
Factors
Primary E18 rat cortical neurons (DIV 13; mixed
culture) were treated with SUB9KITS™ Pten
siRNA-Lipid Nanoparticles (100 ng/mL siRNA)
alone, or SUB9KITS™ Hprt siRNA-Lipid
Nanoparticles (100 ng/mL siRNA) alone, or a
combination
of
both
nanoparticle
preparations. A non-targeted SUB9KITS™
siRNA-Lipid Nanoparticles (100 ng/mL siRNA)
was used as a control. Hprt and Pten mRNA
llevels were quantified 72 h post-treatment
(RT-qPCR).
Hprt
Pten
100
For more information, go to www.sub9kits.com
50
Acknowledgements and Support
0
Control
in vivo
SUB9KITS™ RNA-Lipid Nanoparticles are delivered to the cytoplasm by mimicking
low-density-lipoproteins (LDL) and co-opting the paracrine factor Apolipoprotein
E4 (ApoE4) to mediate cell uptake via the LDL-receptor. Simply adding the
nanoparticles to ApoE-containing primary neuron culture results in >95% uptake
within 4h (data not shown).
SUB9KITS.com
SUB KITS.com
SUB9KITS™ RNA-Lipid Nanoparticles represent a new, highly
effective modality of transfection reagent for loss-offunction and gain-of-function studies in primary neurons in
vitro and in vivo.
siNT
siHprt
siPten
siHprt
siPten
SUB9
SUB9 KITS™ mRNAmRNA-Lipid Nanoparticles Mediate Effective Transfection
and Expression of GFP in Primary Rat Cortical Neuron Cultures.
Cultures.
Precision NanoSystems acknowledges the support of the research groups headed by
Profs. Brian MacVicar and Yu Tian Wang, University of British Columbia.
Primary E18 rat cortical neurons (DIV 13; mixed
culture) were treated with fluorescently-labelled
SUB9KITS™ GFP mRNA-Lipid Nanoparticles (500
ng/mL GFP mRNA). Confocal images were taken
72h post-treatment to confirm nanoparticle
uptake (red, left-hand panel). The right-hand panel
shows an overlay of uptake and GFP expression.
Flow cytometry analysis indicated particle uptake
and subsequent GFP expression in > 90% of
primary neurons (data not shown).
1.888.618.0031 · [email protected]@precision -n an o.com