No abstract

Huntington's disease (HD) is characterized by preferential loss of the medium spiny neurons in the striatum. Using CRISPR/Cas9 and somatic nuclear transfer technology, we established a knockin (KI) pig model of HD that endogenously expresses full-length mutant huntingtin (HTT). By breeding this HD pig model, we have successfully obtained F1 and F2 generation KI pigs. Characterization of founder and F1 KI pigs shows consistent movement, behavioral abnormalities, and early death, which are germline transmittable. More importantly, brains of HD KI pig display striking and selective degeneration of striatal medium spiny neurons. Thus, using a large animal model of HD, we demonstrate for the first time that overt and selective neurodegeneration seen in HD patients can be recapitulated by endogenously expressed mutant proteins in large mammals, a finding that also underscores the importance of using large mammals to investigate the pathogenesis of neurodegenerative diseases and their therapeutics.

CRISPR-Cas9 is a powerful new tool for genome editing, but this technique creates mosaic mutations that affect the efficiency and precision of its ability to edit the genome. Reducing mosaic mutations is particularly important for gene therapy and precision genome editing. Although the mechanisms underlying the CRSIPR/Cas9-mediated mosaic mutations remain elusive, the prolonged expression and activity of Cas9 in embryos could contribute to mosaicism in DNA mutations. Here we report that tagging Cas9 with ubiquitin-proteasomal degradation signals can facilitate the degradation of Cas9 in non-human primate embryos. Using embryo-splitting approach, we found that shortening the half-life of Cas9 in fertilized zygotes reduces mosaic mutations and increases its ability to modify genomes in non-human primate embryos. Also, injection of modified Cas9 in one-cell embryos leads to live monkeys with the targeted gene modifications. Our findings suggest that modifying Cas9 activity can be an effective strategy to enhance precision genome editing.CRISPR-Cas9 combines the type II bacterial clustered, regularly interspaced, palindromic repeats (CRISPR)-associated (Cas) system and a single guide RNA (gRNA), which targets Cas9 to genomic regions complementary to the gRNA 1 . Double-stranded DNA breaks at the targeted locus are generated by Cas9 digestion and repaired by non-homologous end-joining (NHEJ) or homology-directed repair (HDR), resulting in indel mutations [2][3]

The first clinically approved engineered chimeric antigen receptor (CAR) T cell therapies are remarkably effective in a subset of hematological malignancies with few therapeutic options. Although these clinical successes have been exciting, CAR T cells have hit roadblocks in solid tumors that include the lack of highly tumor-specific antigens to target, opening up the possibility of life-threatening “on-target/off-tumor” toxicities, and problems with T cell entry into solid tumor and persistent activity in suppressive tumor microenvironments. Here, we improve the specificity and persistent antitumor activity of therapeutic T cells with synthetic Notch (synNotch) CAR circuits. We identify alkaline phosphatase placental-like 2 (ALPPL2) as a tumor-specific antigen expressed in a spectrum of solid tumors, including mesothelioma and ovarian cancer. ALPPL2 can act as a sole target for CAR therapy or be combined with tumor-associated antigens such as melanoma cell adhesion molecule (MCAM), mesothelin, or human epidermal growth factor receptor 2 (HER2) in synNotch CAR combinatorial antigen circuits. SynNotch CAR T cells display superior control of tumor burden when compared to T cells constitutively expressing a CAR targeting the same antigens in mouse models of human mesothelioma and ovarian cancer. This was achieved by preventing CAR-mediated tonic signaling through synNotch-controlled expression, allowing T cells to maintain a long-lived memory and non-exhausted phenotype. Collectively, we establish ALPPL2 as a clinically viable cell therapy target for multiple solid tumors and demonstrate the multifaceted therapeutic benefits of synNotch CAR T cells.

The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated 9 (Cas9) system is a versatile RNA‐guided mammalian genome modification system. One‐step generation of mouse genome targeting has been achieved by co‐microinjection of one‐cell stage embryos with Cas9 mRNA and small/single guide (sg)RNA. Many studies have focused on enhancing the efficiency of this system. In the present study, we report that simultaneous use of dual sgRNAs to target an individual gene significantly improved the Cas9‐mediated genome targeting with a bi‐allelic modification efficiency of up to 78%. We further observed that the target gene modifications were characterized by efficient germline transmission and site‐dependent off‐target effects, and also that the apolipoprotein E gene knockout‐mediated defects in blood biochemical parameters were recapitulated by CRISPR/Cas9‐mediated heritable gene modification. Our results provide a dual sgRNAs strategy to facilitate CRISPR/Cas9‐mediated mouse genome targeting.