Two-thirds partial hepatectomy (PH) induces differentiated cells in the liver remnant to proliferate and regenerate to its original size. The proliferation-specific HNF-3/fork head homolog-11B protein (HFH-11B; also known as Trident and Win) is a family member of the winged helix/fork head transcription factors and in regenerating liver its expression is reactivated prior to hepatocyte entry into DNA replication (S phase). To examine whether HFH-11B regulates hepatocyte proliferation during liver regeneration, we used the ؊3-kb transthyretin (TTR) promoter to create transgenic mice that displayed ectopic hepatocyte expression of HFH-11B. Liver regeneration studies with the TTR-HFH-11B mice demonstrate that its premature expression resulted in an 8-h acceleration in the onset of hepatocyte DNA replication and mitosis. This liver regeneration phenotype is associated with protracted expression of cyclin D1 and C/EBP␤, which are involved in stimulating DNA replication and premature expression of M phase promoting cyclin B1 and cdc2. Consistent with the early hepatocyte entry into S phase, regenerating transgenic livers exhibited earlier expression of DNA repair genes (XRCC1, mHR21spA, and mHR23B). Furthermore, in nonregenerating transgenic livers, ectopic HFH-11B expression did not elicit abnormal hepatocyte proliferation, a finding consistent with the retention of the HFH-11B transgene protein in the cytoplasm. We found that nuclear translocation of the HFH-11B transgene protein requires mitogenic signalling induced by PH and that its premature availability in regenerating transgenic liver allowed nuclear translocation to occur 8 h earlier than in wild type.The mammalian liver is one of the few adult organs capable of completely regenerating itself in response to cellular injury from toxins, viral infections, or tissue removal (15,38,46). Liver regeneration after two-thirds partial hepatectomy (PH) represents a balance between hepatocyte proliferation and the maintenance of hepatocyte-specific gene expression required for liver homeostasis (22,46). A potent activation of hepatocyte immediate early transcription factors is observed during liver regeneration and includes c-Jun, c-Fos, c-Myc, NF-B, signal transducers, and activators of transcription 3 (stat3) and the CCAAT/enhancer protein ␤ (C/EBP␤) genes (7,9,25,46). Furthermore, maintenance of hepatocyte-specific gene transcription is coincident with sustained expression of hepatocyte nuclear factor genes (16,20,41). More recent genetic data demonstrated that the cytokine interleukin-6 (IL-6) plays an important role in establishing responsiveness of hepatocytes to growth factors which are released after liver injury (8,54). In a PH model of liver regeneration, homozygous null interleukin-6 (IL-6) or type 1 tumor necrosis factor receptor (TNFR-I) mice exhibited a 70% reduction in hepatocyte replication and this proliferation defect was eliminated by an intraperitoneal injection of IL-6 prior to surgery (8,43,54). This proliferation defect was accompanied by a failur...

The winged helix transcription factor, hepatocyte nuclear factor-3␤ (HNF-3␤), mediates the hepatocytespecific transcription of numerous genes important for liver function. However, the in vivo role of HNF-3␤ in regulating these genes remains unknown because homozygous null HNF3␤ mouse embryos die in utero prior to liver formation. In order to examine the regulatory function of HNF-3␤, we created transgenic mice in which the ؊3-kb transthyretin promoter functions to increase hepatocyte expression of the rat HNF-3␤ protein.Postnatal transgenic mice exhibit growth retardation, depletion of hepatocyte glycogen storage, and elevated levels of bile acids in serum. The retarded growth phenotype is likely due to a 20-fold increase in hepatic expression of insulin-like growth factor binding protein 1 (IGFBP-1), which results in elevated levels in serum of IGFBP-1 and limits the biological availability of IGFs required for postnatal growth. The defects in glycogen storage and serum bile acids coincide with diminished postnatal expression of hepatocyte genes involved in gluconeogenesis (phosphoenolpyruvate carboxykinase and glycogen synthase) and sinusoidal bile acid uptake (Ntcp), respectively. These changes in gene transcription may result from the disruptive effect of HNF-3␤ on the hepatic expression of the endogenous mouse HNF-3␣,-3␤, -3␥, and -6 transcription factors. Furthermore, adult transgenic livers lack expression of the canalicular phospholipid transporter, mdr2, which is consistent with ultrastructure evidence of damage to transgenic hepatocytes and bile canaliculi. These transgenic studies represent the first in vivo demonstration that the HNF-3␤ transcriptional network regulates expression of hepatocyte-specific genes required for bile acid and glucose homeostasis, as well as postnatal growth.The liver performs essential functions in the body by uniquely expressing both hepatocyte-specific genes encoding plasma proteins and enzymes involved in the detoxification and in the homeostasis of glucose, cholesterol, and bile salts (4). Functional analysis of numerous hepatocyte-specific promoter and enhancer regions reveals that they are composed of multiple cis-acting DNA sequences that bind different families of hepatocyte nuclear factors (HNF) (reviewed in reference 4). These include the HNF-1, HNF-3, HNF-4, CCAAT/enhancer binding protein (C/EBP), HNF-6, and fetoprotein transcription factor families (4,29,15,52,53,57). Although none of these transcriptional regulatory proteins is entirely liver specific, the requirement for combinatorial protein interactions among them in order to achieve abundant transcriptional activity plays an important role in maintaining hepatocyte-specific gene expression.The HNF-3 proteins are members of an extensive family of transcription factors that share homology in the winged helix DNA binding domain and use a modified helix-turn-helix motif to bind DNA as a monomer (8, 37). To date, the winged helix family consists of over 50 members, which play important roles in the differentiat...

Transient delivery of CRISPR-based genome editing effectors is important to reduce off-target effects and immune responses. Recently extracellular vesicles (EVs) have been explored for Cas9 ribonucleoprotein (RNP) delivery. However, lack of mechanisms to enrich RNPs into EVs limited the efficiency of EVs as a RNP delivery vehicle. Here we describe a mechanism to actively enrich RNPs into EVs. We used the specific interaction between RNA aptamer and aptamer-binding protein (ABP) to enrich RNPs into EVs. We inserted RNA aptamer com into single guide RNA (sgRNA), and fused com-binding ABP Com to both termini of tetraspan protein CD63 that is abundant in exosomes. We found that the Com/com interaction enriched Cas9 and adenine base editor (ABE) RNPs into EVs, via forming a three-component complex including CD63-Com fusion protein, com-modified sgRNA and Cas9 or ABE. The RNP enriched EVs are efficient in genome editing and transiently expressed. The system is capable of delivering RNPs targeting multiple loci for multiplex genome editing. In addition, Cas9 from different species can be used together. The EV-delivered RNPs are active in vivo. The data show that the aptamer and ABP interactions can be utilized to actively enrich RNPs into EVs for improved genome editing efficiency and safety. K E Y WO R D S adenine base editor, aptamer, aptamer-binding protein, CD63, CRISPR/Cas9, delivery, extracellular vesicle, ribonucleoprotein  INTRODUCTION Extracellular vesicles (EVs) can be broadly divided into two main categories, exosomes and microvesicles, based on the mechanisms of generation. Exosomes are heterogeneous membranous vesicles released by various cells via inward budding of multivesicular bodies and subsequent fusion of the multivesicular body membranes with the plasma membrane (Heijnen et al., 1999). Exosomes play important roles in intercellular crosstalk and disease pathogenesis, and are believed to function by transporting RNAs, proteins and lipids from one cell to the other (Ratajczak et al., 2006). Microvesicles are generated by the outward budding and fission of the plasma membrane and the subsequent release of vesicles into the extracellular space. Exosomes and microvesicles overlap in sizes and currently it is difficult to separate the two types of vesicles in preparation. The transportation capability of EVs prompted the exploration of using EVs as drug delivery vehicles (Alvarez-Erviti et al.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system discovered using bacteria has been repurposed for genome editing in human cells. Transient expression of the editor proteins (e.g. Cas9 protein) is desirable to reduce the risk of mutagenesis from off-target activity. Using the specific interaction between bacteriophage RNA-binding proteins and their RNA aptamers, we developed a system able to package up to 100 copies of Staphylococcus aureus Cas9 ( SaCas9 ) mRNA in each l enti v irus- l ike bionano p article (LVLP). The SaCas9 LVLPs mediated transient SaCas9 expression and achieved highly efficient genome editing in the presence of guide RNA. Lower off-target rates occurred in cells transduced with LVLPs containing SaCas9 mRNA, compared with cells transduced with adeno-associated virus or lentivirus expressing SaCas9 . Our LVLP system may be useful for efficiently delivering Cas9 mRNA to cell lines and primary cells for in vitro and in vivo gene editing applications.