siRNA-optimized Modifications for Enhanced In Vivo Activity - PubMed (original) (raw)
siRNA-optimized Modifications for Enhanced In Vivo Activity
Denise M Kenski et al. Mol Ther Nucleic Acids. 2012.
Abstract
Current modifications used in small interfering RNAs (siRNAs), such as 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F), improve stability, specificity or immunogenic properties but do not improve potency. These modifications were previously designed for use in antisense and not siRNA. We show, for the first time, that the siRNA-optimized novel 2'-O modifications, 2'-O-benzyl, and 2'-O-methyl-4-pyridine (2'-O-CH2Py(4)), are tolerated at multiple positions on the guide strand of siRNA sequences in vivo. 2'-O-benzyl and 2'-O-CH2Py(4) modifications were tested at each position individually along the guide strand in five sequences to determine positions that tolerated the modifications. The positions were combined together and found to increase potency and duration of siRNAs in vivo compared to their unmodified counterparts when delivered using lipid nanoparticles. For 2'-O-benzyl, four incorporations were tolerated with similar activity to the unmodified siRNA in vivo, while for 2'-O-CH2Py(4) six incorporations were tolerated. Increased in vivo activity was observed when the modifications were combined at positions 8 and 15 on the guide strand. Understanding the optimal placement of siRNA-optimized modifications needed for maximal in vivo activity is necessary for development of RNA-based therapeutics.
Figures
Figure 1
In vitro activity of 2′-o-benzyl and 2′-o-CH2Py (4). (a) Structure of the nucleotides used for oligonucleotide synthesis with 2′-OH or 2′-O-benzyl (in red). Adenosine is representative of the modifications to the other bases, guanosine, cytosine and uracil. (b, c) mRNA degradation of target as determined by quantitative PCR (qPCR) and compared to unmodified. Values are shown as log 2 deviations from unmodified. A log ratio of 1 represents a twofold change. Each of the five small interfering RNA (siRNA) sequences, represented by a different colored dot, contain complementary all ribose guide and passenger strands and were transfected into Hep1-6 cells at 10 nmol/l. The median value at each position is represented by a black bar. The dotted lines represent the experimental variation generated from the qPCR. Individual (b) 2′-O-benzyl or (c) 2′-O-CH2Py(4) were substituted at the indicated position on the guide strand of the duplex. Activity of the chemical unmodified (all ribose) siRNA is at the zero line. Increased mRNA degradation activity compared to an unmodified siRNA would result in a ratio higher than zero and would be advantageous to the siRNA and all points below the zero line have decreased activity.
Figure 2
2′-O-benzyls demonstrate enhanced in vivo activity after intravenous (i.v.) injection. (a) Unmodified ApoB(10162) small interfering RNA (siRNA), siRNAs with an 2′-O-benzyl at either position 5, 8, 15, or 19 were formulated into lipid nanoparticles and injected in mice at 3 mg/kg. The livers were harvested and ApoB mRNA levels were evaluated by quantitative PCR (qPCR) 72 hours after injection (* = student′s t-test, P < 0.05). Each dot represents an individual mouse and bars represent the mean and standard deviation of all mice per group. (b) Unmodified ApoB(10162) siRNA, siRNAs with an 2′-O-benzyl at either position 5, 8, 15, or 19 were formulated into lipid nanoparticles and injected in mice at 3 mg/kg. The livers were harvested and apolipoprotein B (ApoB) mRNA levels were evaluated by qPCR at 3, 7, and 14 days after injection. Data are presented as the mean and s.e.m. *P < 0.05 versus unmodified; **P < 0.005 versus unmodified.
Figure 3
In vivo evaluation of 2′-O-benzyls at positions 8 and 15 on the small interfering RNA (siRNA) guide strand. (a) Unmodified ApoB(10162), (b) ApoB(9514), and (c) Luc(80) siRNAs and the same siRNAs with 2′-O-benzyls at positions 8 and 15 were formulated into lipid nanoparticles and injected in mice at 3 mg/kg. The livers were harvested for Apo(9514) and ApoB(10162) mice and mRNA levels were evaluated by quantitative PCR (qPCR) at 3 and 14 days after injection. Luciferase mice were imaged every other day after dosing. Area under the curve was calculated (d) for Luc(80) siRNAs as a measure of duration. Data are presented as the mean and s.e.m. *P < 0.05 versus unmodified; **P < 0.005 versus unmodified; ***P < 0.0005 versus unmodified.
Figure 4
In vivo evaluation of 2′-O-benzyls at positions 5, 8, 15, and 19 on the small interfering RNA (siRNA) guide strand. Unmodified (a) ApoB(10162), (b) ApoB(9514), and (c) Luc(80) siRNAs and the same siRNAs with 2′-O-benzyls at positions 8 and 15 were formulated into lipid nanoparticles and injected in mice at 3 mg/kg. The livers were harvested for Apo(9514) and ApoB(10162) mice and mRNA levels were evaluated by quantitative PCR (qPCR) at 3 and 14 days after injection. Luciferase mice were imaged every other day after dosing. Area under the curve was calculated (d) for Luc(80) siRNAs as a measure of duration. Data are presented as the mean and s.e.m. *P < 0.05 versus unmodified; **P < 0.005 versus unmodified; ***P < 0.0005 versus unmodified.
Figure 5
In vivo duration of 2′-O-CH2Py(4) on the small interfering RNA (siRNA) guide strand. Unmodified ApoB(10162) siRNA and the same siRNAs with 2′-O-CH2Py(4) at positions (a) 8 and 15 or (b) 5, 6, 8, 10, 15, and 19 on the guide strand were formulated into lipid nanoparticles and injected in mice at 3 mg/kg. The livers were harvested and mRNA levels were evaluated by quantitative PCR (qPCR) at 3 and 14 days after injection. Data are presented as the mean and s.e.m. *P < 0.05 versus unmodified.
References
- Schwarz DS, Hutvágner G, Du T, Xu Z, Aronin N., and, Zamore PD. Asymmetry in the assembly of the RNAi enzyme complex. Cell. 2003;115:199–208. - PubMed
- Schwarz DS, Hutvágner G, Haley B., and, Zamore PD. Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways. Mol Cell. 2002;10:537–548. - PubMed
- Rand TA, Petersen S, Du F., and, Wang X. Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation. Cell. 2005;123:621–629. - PubMed
LinkOut - more resources
Full Text Sources
Other Literature Sources