Coordinated alterations in RNA splicing and epigenetic regulation drive leukaemogenesis.

Transcription and pre-mRNA splicing are key steps in the control of gene expression and mutations in genes regulating each of these processes are common in leukaemia1,2. Despite the frequent overlap of mutations affecting epigenetic regulation and splicing in leukaemia, how these processes influence one another to promote leukaemogenesis is not understood and, to our knowledge, there is no functional evidence that mutations in RNA splicing factors initiate leukaemia. Here, through analyses of transcriptomes from 982 patients with acute myeloid leukaemia, we identified frequent overlap of mutations in IDH2 and SRSF2 that together promote leukaemogenesis through coordinated effects on the epigenome and RNA splicing. Whereas mutations in either IDH2 or SRSF2 imparted distinct splicing changes, co-expression of mutant IDH2 altered the splicing effects of mutant SRSF2 and resulted in more profound splicing changes than either mutation alone. Consistent with this, co-expression of mutant IDH2 and SRSF2 resulted in lethal myelodysplasia with proliferative features in vivo and enhanced self-renewal in a manner not observed with either mutation alone. IDH2 and SRSF2 double-mutant cells exhibited aberrant splicing and reduced expression of INTS3, a member of the integrator complex3, concordant with increased stalling of RNA polymerase II (RNAPII). Aberrant INTS3 splicing contributed to leukaemogenesis in concert with mutant IDH2 and was dependent on mutant SRSF2 binding to cis elements in INTS3 mRNA and increased DNA methylation of INTS3. These data identify a pathogenic crosstalk between altered epigenetic state and splicing in a subset of leukaemias, provide functional evidence that mutations in splicing factors drive myeloid malignancy development, and identify spliceosomal changes as a mediator of IDH2-mutant leukaemogenesis.

Kupffer cell release of platelet activating factor drives dose limiting toxicities of nucleic acid nanocarriers.

This work establishes that Kupffer cell release of platelet activating factor (PAF), a lipidic molecule with pro-inflammatory and vasoactive signaling properties, dictates dose-limiting siRNA nanocarrier-associated toxicities. High-dose intravenous injection of siRNA-polymer nano-polyplexes (si-NPs) elicited acute, shock-like symptoms in mice, associated with increased plasma PAF and consequently reduced PAF acetylhydrolase (PAF-AH) activity. These symptoms were completely prevented by prophylactic PAF receptor inhibition or Kupffer cell depletion. Assessment of varied si-NP chemistries confirmed that toxicity level correlated to relative uptake of the carrier by liver Kupffer cells and that this toxicity mechanism is dependent on carrier endosome disruptive function. 4T1 tumor-bearing mice, which exhibit increased circulating leukocytes, displayed greater sensitivity to these toxicities. PAF-mediated toxicities were generalizable to commercial delivery reagent in vivo-jetPEI® and an MC3 lipid formulation matched to an FDA-approved nanomedicine. These collective results establish Kupffer cell release of PAF as a key mediator of siRNA nanocarrier toxicity and identify PAFR inhibition as an effective strategy to increase siRNA nanocarrier tolerated dose.

Dual carrier-cargo hydrophobization and charge ratio optimization improve the systemic circulation and safety of zwitterionic nano-polyplexes.

While polymeric nano-formulations for RNAi therapeutics hold great promise for molecularly-targeted, personalized medicine, they possess significant systemic delivery challenges including rapid clearance from circulation and the potential for carrier-associated toxicity due to cationic polymer or lipid components. Herein, we evaluated the in vivo pharmacokinetic and safety impact of often-overlooked formulation parameters, including the ratio of carrier polymer to cargo siRNA and hydrophobic siRNA modification in combination with hydrophobic polymer components (dual hydrophobization). For these studies, we used nano-polyplexes (NPs) with well-shielded, zwitterionic coronas, resulting in various NP formulations of equivalent hydrodynamic size and neutral surface charge regardless of charge ratio. Doubling nano-polyplex charge ratio from 10 to 20 increased circulation half-life five-fold and pharmacokinetic area under the curve four-fold, but was also associated with increased liver enzymes, a marker of hepatic damage. Dual hydrophobization achieved by formulating NPs with palmitic acid-modified siRNA (siPA-NPs) both reduced the amount of carrier polymer required to achieve optimal pharmacokinetic profiles and abrogated liver toxicities. We also show that optimized zwitterionic siPA-NPs are well-tolerated upon long-term, repeated administration in mice and exhibit greater than two-fold increased uptake in orthotopic MDA-MB-231 xenografts compared to commercial transfection reagent, in vivo-jetPEI®. These data suggest that charge ratio optimization has important in vivo implications and that dual hydrophobization strategies can be used to maximize both NP circulation time and safety.