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. 2019 Mar 7;27(4):866–877. doi: 10.1016/j.ymthe.2019.03.003

Figure 2.

Figure 2

Optimization of mRNA Construct and Animal Pretreatment

(A) Natural and modified nucleosides used during the in vitro transcription reactions to create unmodified and modified mRNA, respectively. (B) Liver genome-editing results from mice injected with a single 2 mg/kg dose of LNP containing 48641/31523 ZFN mRNAs (n = 8 per group) (p = 0.067). (C) Genome modification results following a single administration of 0.5 mg/kg LNP comprising 25% pU substituted or unmodified mRNAs encoding ZFNs 48641 and 31523 (n = 5–6 per group) (p = 0.0029). (D) Genome-editing results from repeat dosing (14-day intervals) of 2 mg/kg LNP containing 48641/31523 ZFN mRNAs with 25% pU nucleoside substitution (open circles) or no nucleoside substitution (closed circles) (n = 8 per group) (p = 0.000016 comparing three doses of ARCA versus Cap1). (E) Repeat dosing (14-day intervals, 0.5 mg/kg) of LNP containing 48641/31523 ZFN mRNAs containing different poly(A) lengths and composition of uridines in the protein coding sequence as indicated. Livers were harvested 7 days post-dosing. “64polyA,” “128polyA,” and “193polyA” refer, respectively, to 64, 128, or 193 long poly(A) regions while “uridine-depleted” refers to polynucleotides with at a percentage of uridines deleted from the wobble positions in the codons (n = 3 per group) (p = 0.089 comparing 1 dose of original versus uridine-depleted 193poly(A)); (n = 3–6 per group) (p = 0.021 comparing one dose of original versus uridine-depleted 64poly(A)); (n = 3–6 per group) (p = 0.085 comparing three doses of original 64poly(A) versus 193poly(A)). (F) Single dose of LNP containing 48641/31523 ZFN mRNAs, purified either via silica column or HPLC and injected into mice at 1 mg/kg and livers harvested 28 days post-dosing. (G) Genome-editing results from repeat dosing (14-day intervals, 2 mg/kg) of LNP containing 48641/31523 ZFN mRNAs (n = 3 per group) (p = 0.075 comparing 3 doses with and without Dex). (H) Genome-modification results following a single administration 0.5 mg/kg LNP containing 25% pU substituted 48641/31523 ZFN mRNAs into mice which were either fed ad libitum or mice that were fasted overnight prior to LNP dosing (n = 4 per group) (p = 0.012). (I) Genome-modification results following a single administration of 0.5 mg/kg LNP containing 25% pU substituted 48641/31523 ZFN mRNAs into either male or female C57BL6 wild-type mice (n = 4 females and 3 males) (p = 0.0047).