STICKY SIRNA™ from Polyplus-transfection

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STICKY SIRNA™

The STICKY SIRNA™ are a new class of small interfering RNAs designed to improve in vivo siRNA delivery. The technology involves extending the opposite ends of interfering RNAs with short complementary A(5-8)/T(5-8) 3′ sequences able to form concatemers in the presence of a cationic polymer such as in vivo-jetPEI™ and thus form complexes as stable as with genes. With this new technology, siRNA remain associated with the delivery agent and are able to induce RNA interference in vivo. This innovation is applicable to therapeutic siRNAs for a wide variety of pathologies.

Scheme I : STICKY SIRNA™ concept: concatemerization of siRNA by their overhangs to mimic long double stranded RNA

STICKY SIRNA™ advantages

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Increased complexes stability compared to standard siRNA to improve delivery

Making siRNA « looking like » a gene, via short complementary A(5-8)/T(5-8) overhangs, increases complex stability.
As can be seen in figure 1, complexes of STICKY SIRNA™ capable of forming non covalent concatemers via (dA)8•(dT)8 bridges become resistant to exchange in presence of heparan sulfate (large polymeric anion found at the cell surface).

Figure 1. Gel electrophoresis of siRNA/jetPEI™ complexes incubated with increasing amounts of heparan sulfate (HS). Because of aggregation, siRNA/jetPEI™ complexes remain in the well (0 eq of HS). Incubation for 30 min with 20-40 (wt/wt) eq HS releases siRNA (left and right), but not STICKY SIRNA™ (center), from the complexes.

Improved silencing efficiency in vitro

The effect of concatemer formation on gene silencing efficiency was compared between sticky (dA)5-siRNA-(dT)5 and standard (dT)2-siRNA-(dT)2, using the GL3 luciferase gene stably expressed by A549 epithelial cells.

Sticky small interfering RNAs enhance gene silencing in vitro up to tenfold when complexed with in vivo-jetPEI™ compared to standard siRNA.

Figure 2. Endogeneous luciferase silencing with STICKY SIRNA™ complexed with in vivo-jetPEI™. A549-GL3Luc cells were incubated with (dA)5-siRNA-(dT)5 and standard (dT)2-siRNA-(dT)2 complexed with in vivo-jetPEI™ (N/P=5). Luciferase silencing efficiency was determined after 48 h.

No pro-inflammatory cytokine or hepatic enzyme production upon i.v. injection

No induction of pro-inflammatory cytokines was detected after delivery of STICKY SIRNA™ complexed with in vivo-jetPEI™ (figure 3). Moreove, there was no induction of hepatic enzymes (ALAT, ASAT, LDH and ALP) after STICKY SIRNA™ delivery with in vivo-jetPEI™ (Bonnet et al, 2008).

	siRNA	STICKY SIRNA™	siRNA/ in vivo-jetPEI™	STICKY SIRNA/ in vivo-jetPEI™	LPS
TNF-α (1h)	< 4	< 4	< 4	< 4	4425 ± 2391
IFN-γ (6h)	< 15	< 15	< 15	< 15	4952 ± 9905
IL-12/IL-23 (3h)	437 ± 606	253 ± 242	1301 ± 817	732 ± 547	23932 ± 9411
IL-6 (3h)	58 ± 122	153 ± 166	1204 ± 985	1306 ± 754	24762 ± 27614
IL1-β (6h)	< 8	< 8	< 8	< 8	ND

Figure 3. Pro-inflammatory cytokines levels measured after siRNA or STICKY SIRNA™ /in vivo-jetPEI™ delivery.
siRNA or STICKY SIRNA™ (40 µg) complexed (N/P=8) or not with in vivo-jetPEI™ in 5% glucose solution were injected intravenously through retro-orbital sinus. Blood was collected and level of pro-inflammatory cytokines was evaluated by ELISA (at 1h for TNF-α dosage, 6h for IFN-γ, 3h for IL-12/IL-23 and IL-6 and 6h for IL-1β). As a positive control, we injected E. Coli LPS (50 µg in 1 mL PBS 1x) intraperitonealy. Each value represents the mean ± SD (n=8 for specific assays and n=4 for LPS). Concentrations are expressed in pg/ml.

Tested successfully in vivo: inhibition of PC-3 tumor metastasis

Intraperitoneal injection of STICKY SIRNA™ cyclinB1 in mice, in a PC-3 tumor model, leads to an inhibition of tumor metastasis compared to mismatch and control. Moreover, mice survival is increased dramatically with STICKY SIRNA™ delivered by in vivo-jetPEI™.

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