Lipofectamine

Example 17

To further validate the activity of the DMPK siRNAs, many of the sequences that showed the best activity in the initial screen were selected for a follow-up evaluation in dose response format. Once again, two human cell lines were used to assess the in vitro activity of the DMPK siRNAs: first, SJCRH30 human rhabdomyosarcoma cell line; and second, Myotonic Dystrophy Type 1 (DM1) patient-derived immortalized human skeletal myoblasts. The selected siRNAs were transfected in a 10-fold dose response at 100, 10, 1, 0.1, 0.01, 0,001, and 0.0001 nM final concentrations or in a 9-fold dose response at 50, 5.55556, 0.617284, 0.068587, 0.007621, 0.000847, and 0.000094 nM final concentrations. The siRNAs were formulated with transfection reagent Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer's “forward transfection” instructions. Cells were plated 24 h prior to transfection in triplicate on 96-well tissue culture plates, with 8500 cells per well for SJCRH30 and 4000 cells per well for DM1 myoblasts. At 48 h (SJCRH30) or 72 h (DM1 myoblasts) post-transfection cells were washed with PBS and harvested with TRIzol® reagent (Life Technologies). RNA was isolated using the Direct-zol-96 RNA Kit (Zymo Research) according to the manufacturer's instructions. 10 μl of RNA was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. cDNA samples were evaluated by qPCR with DMPK-specific and PPIB-specific TaqMan human gene expression probes (Thermo Fisher) using TaqMan® Fast Advanced Master Mix (Applied Biosystems). DMPK values were normalized within each sample to PPIB gene expression. The quantification of DMPK downregulation was performed using the standard 2^−ΔΔCt a method. All experiments were performed in triplicate, with Tables 16A-B, 17A-B, and 18A-B presenting the mean values of the triplicates as well as the calculated IC₅₀ values determined from fitting curves to the dose-response data by non-linear regression.

Example 2

iPS cells were prepared according to protocols known in the art and seeded in a Geltrex®-Matrix coated 12-well culture dish. Transfection was performed in iPSCs with 3 ul of Lipofectamine® 2000 or 3 ul of Lipofectamine® 3000 as indicated and according to manufacturer's instructions, to deliver a GeneArt® CRISPR Nuclease vector targeting the HPRT locus. Transfection was also performed with GeneArt® CRISPR Nuclease RNA editing system targeting the HPRT locus and 3 ul of Formulation 21 lipid aggregate complex. RNA editing system utilizes a Cas9 mRNA, which was prepared via in vitro transcription with the Ambion® mMESSAGE mMACHINE® Kit, and a gRNA which was transcribed using the Ambion® MEGAshortscript™ Kit. Cells were harvested 72-hours post-transfection and cleavage efficiency was determined using the GeneArt® Genomic Cleavage Detection Kit.

Results are shown in FIG. 2A and FIG. 2B, which clearly demonstrate that using an mRNA based form of Cas9 with a guide-RNA for gene editing with the lipid aggregates described herein for transfection results in at least 4-fold more targeted cleavage of the host cell genome when compared to standard DNA based editing approaches.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

Example 7

ncRNA performance can be modified by adding a cap structure to the 5′ end and/or adding a poly(A) tail at the 3′ end, as depicted in FIG. 55. As shown in FIG. 56, using ncRNA with either or both protection by cap and tail lowered indels as compared uncapped/untailed ncRNAs. In this experiment, a 4 component all-RNA system (RT mRNA+Cas9 mRNA+ncRNA-sgRNA+sgRNA) was delivered to HEK293T cells by Lipofectamine MessengerMAX. All RNA was transfected at a fixed amount RT mRNA 100 ng, ncRNA-sgRNA 400 ng, Cas9 mRNA 100 ng, and sgRNA 5 ng. ncRNA-gRNA fusion was either capped (+cap −tail) or poly-A tailed (−cap +tail) or both capped and poly-A tailed (+cap +tail). Using RNA without end protection (−cap −tail) produced ˜4.5% precise edits and the editing was dependent on retron since the absence of RT abrogated precise editing. Using RNA with either or both protection by cap and tail produced lower precise editing (left graph) but lowered indels (right graph) than without cap and tail.