1. Volume 20, Issue 2, Pages 356-366 (February 2012)
Atelocollagen-mediated Systemic Delivery Prevents Immunostimulatory Adverse Effects of siRNA in Mammals 
Shinichiro Inaba, Shunji Nagahara, Naoki Makita, Yuzo Tarumi, Takuji Ishimoto, Seiichi Matsuo, Kenji Kadomatsu, Yoshifumi Takei 
Molecular Therapy 
Volume 20, Issue 2, Pages (February 2012)
DOI: /mt
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
isRNA mixed with atelocollagen did not induce any type-I IFNs in mice. (a) Structures of isRNA harboring the interferon (IFN)-inducible sequence (5′-UGUGU-3′). (b) Balb/c mice were intravenously injected with isRNA alone, atelocollagen alone, isRNA with atelocollagen, DOTAP alone, isRNA with DOTAP, In vivo JET-PEI alone, or isRNA with In vivo JET-PEI, respectively. The dose of isRNA was fixed at 50 µg per mouse. The levels of both IFN-α and IFN-β in mouse serum (6 hours postinjection) were evaluated using each specific ELISA. The results represent the means ± SD (n = 4). N.D., not detected. (c) Different mouse strains were examined. C57BL/6J, Balb/c, and ICR were intravenously injected with isRNA mixed with either atelocollagen or DOTAP as in b. The levels of both IFN-α and IFN-β in mouse serum (6 hours postinjection) were evaluated. The results represent the means ± SD (n = 4). N.D., not detected. ELISA, enzyme-linked immunosorbent assay; isRNA, immunostimulatory RNA.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
isRNA mixed with Invivofectamine but not with atelocollagen potently induced type-I IFNs and an inflammatory cytokine, TNF-α, in mice. (a,b) Balb/c mice were intravenously injected with various reagents as indicated in the figures. The dose of isRNA was fixed at 50 µg per mouse. The levels of (a) IFN-α, IFN-β and (b) TNF-α were evaluated at 6 hours following the injection using each specific ELISA. The results represent the means ± SD (n = 4). (c) The relationship between isRNA dose and IFN-response in mice. isRNA (12.5, 25, or 50 µg) mixed with Invivofectamine was intravenously injected into mice. First, the isRNA/Invivofectamine complex (50 µg) was prepared, and then it was diluted with 5% glucose. Each IFN-α level in mouse serum was evaluated at 6 hours following the injection as in a. The results represent the means ± SD (n = 4). (d) Time-course experiments. Each IFN-α level in mouse serum at 3, 6, 12, and 24 hours postinjection was determined by the specific ELISA. Two kinds of formulations (isRNA with Invivofectamine and isRNA with atelocollagen) were examined. The results represent the means ± SD (n = 4). N.D., not detected. ELISA, enzyme-linked immunosorbent assay; IFN, interferon; isRNA; immunostimulatory RNA; TNF, tumor necrosis factor.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
isRNA with Invivofectamine but not with atelocollagen induced secretion of IFN-α from whole blood. (a) The experimental procedures, including information on the mixing of test reagents. A dose of isRNA was fixed at 50 µg/35 mm dish. The information on the test reagents was provided in the box. Each test reagent was mixed with whole blood from a (b) mouse or a (c) human. (b,c) Each sample was collected at 6 or 24 hours after the mixing, and the serum fraction was separated by centrifugation. The level of IFN-α was determined by each specific ELISA. The results represent the means ± SD (n = 3). ELISA, enzyme-linked immunosorbent assay; IFN, interferon; isRNA; immunostimulatory RNA.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
isRNA with Invivofectamine but not with atelocollagen induced IFN-α and inflammatory cytokines in human PBMCs in vitro. (a,b) Human PBMCs were treated with isRNA alone, atelocollagen alone, isRNA with atelocollagen, Invivofectamine alone, or isRNA with Invivofectamine, respectively. The isRNA concentration was kept constant at 100 nmol/l. Twenty-four hours later, the culture supernatants were collected by centrifugation. The secretion levels of (a) IFN-α, or (b) TNF-α, IL-1β, IL-6, and IL-12 were determined. The results represent the means ± SD (n = 4). IFN, interferon; isRNA; immunostimulatory RNA; PBMC, peripheral blood mononuclear cell.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
The expression levels of IFN-response genes in human PBMCs treated with isRNA with atelocollagen or Invivofectamine. (a,b) Human PBMCs were treated with isRNA with atelocollagen or Invivofectamine. At the time point of 3, 6, or 24 hours after the treatment, each total RNA was extracted. All of the total RNA were processed for quantitative real-time RT-PCR to determine the expression of (a) OAS1 and OAS2, (b) IRF9 and IFIT1. 18S rRNA was used as a control. The results represent the means ± SD (n = 4). IFN, interferon; isRNA; immunostimulatory RNA; PBMC, peripheral blood mononuclear cell; rRNA, ribosomal RNA; RT-PCR, reverse transcriptase-PCR.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
isRNA with Invivofectamine but not with atelocollagen was incorporated into human PBMCs in vitro. (a) Human PBMCs were treated with the isRNA/atelocollagen or isRNA/Invivofectamine complex. Six hours later, the cells were collected by centrifugation, and each total RNA was extracted. The amount of isRNA was determined by hybridizing with sequence-specific fluorescence-labeled oligoribonucleotide probes, followed by quantification with reversed-phase HPLC. The results represent the means ± SD (n = 4). N.D., not detected. (b) Human PBMCs were treated with the Cy3-labeled isRNA/atelocollagen, or Cy3-labeled isRNA/Invivofectamine complex. All of the cells were photographed using a confocal microscope system (Nikon A1 Rsi; Nikon, Tokyo, Japan). Hoechst was used for nuclear staining. Differential interference contrast (DIC) images were also obtained, and then merged with the fluorescence images. Bars, 10 µm. (c) Three-dimensional images were constructed using the NIS-Elements C software package (Nikon) according to the instruction manual. HPLC, high-performance liquid chromatography; isRNA; immunostimulatory RNA; PBMC, peripheral blood mononuclear cell.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
Therapeutic effect of MCP-1 siRNA mixed with four different delivery vehicles on the CHS model. (a) The therapeutic procedures in the CHS model targeting MCP-1. Sensitized mice were intravenously injected with MCP-1 siRNA or MCP-1 siRNA-SCR mixed with various delivery vehicles (atelocollagen, Invivofectamine, In vivo JET-PEI, or DOTAP). Mice were treated with two injections (30 minutes and 8 hours) after the ear challenge. The dose of the siRNA was constantly fixed at 50 µg/injection. Ear swelling was determined 24 hours after the ear challenge, and the ears were collected (sacrificed). (b) The results of ear swelling. The results represent percent ratios toward no treatment (NT). The results also represent the means ± SE (n = 6). *P < (c) The excised ears at 24 hours after the ear challenge were homogenized and then centrifuged. The amount of MCP-1 in each supernatant was measured by a specific ELISA for mouse MCP-1. The results represent the means ± SE (n = 6). *P < CHS, contact hypersensitivity; ELISA, enzyme-linked immunosorbent assay; MCP-1, monocyte chemoattractant protein-1; siRNA, short interfering RNA.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions