Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo.

Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs for segmental deletions, or a single sgRNA with a homology-directed repair (HDR) template. We also use anti-CRISPR proteins to enable production of vectors that self-inactivate via Nme2Cas9 cleavage. We further introduce a nanopore-based sequencing platform that is designed to profile rAAV genomes and serves as a quality control measure for vector homogeneity. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice by HDR-based correction of the disease allele. These results will enable the engineering of single-vector AAVs that can achieve diverse therapeutic genome editing outcomes.

YAP1 Withdrawal in Hepatoblastoma Drives Therapeutic Differentiation of Tumor Cells to Functional Hepatocyte-Like Cells.

BACKGROUND AND AIMS: Despite surgical and chemotherapeutic advances, the 5-year survival rate for stage IV hepatoblastoma (HB), the predominant pediatric liver tumor, remains at 27%. Yes-associated protein 1 (YAP1) and ß-catenin co-activation occurs in 80% of children's HB; however, a lack of conditional genetic models precludes tumor maintenance exploration. Thus, the need for a targeted therapy remains unmet. Given the predominance of YAP1 and ß-catenin activation in HB, we sought to evaluate YAP1 as a therapeutic target in HB. APPROACH AND RESULTS: We engineered the conditional HB murine model using hydrodynamic injection to deliver transposon plasmids encoding inducible YAP1S127A , constitutive ß-cateninDelN90 , and a luciferase reporter to murine liver. Tumor regression was evaluated using bioluminescent imaging, tumor landscape characterized using RNA and ATAC sequencing, and DNA footprinting. Here we show that YAP1S127A withdrawal mediates more than 90% tumor regression with survival for 230+ days in mice. YAP1S127A withdrawal promotes apoptosis in a subset of tumor cells, and in remaining cells induces a cell fate switch that drives therapeutic differentiation of HB tumors into Ki-67-negative hepatocyte-like HB cells ("HbHeps") with hepatocyte-like morphology and mature hepatocyte gene expression. YAP1S127A withdrawal drives the formation of hbHeps by modulating liver differentiation transcription factor occupancy. Indeed, tumor-derived hbHeps, consistent with their reprogrammed transcriptional landscape, regain partial hepatocyte function and rescue liver damage in mice. CONCLUSIONS: YAP1S127A withdrawal, without silencing oncogenic ß-catenin, significantly regresses hepatoblastoma, providing in vivo data to support YAP1 as a therapeutic target for HB. YAP1S127A withdrawal alone sufficiently drives long-term regression in HB, as it promotes cell death in a subset of tumor cells and modulates transcription factor occupancy to reverse the fate of residual tumor cells to mimic functional hepatocytes.

Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope.

CRISPR-Cas9-associated base editing is a promising tool to correct pathogenic single nucleotide mutations in research or therapeutic settings. Efficient base editing requires cellular exposure to levels of base editors that can be difficult to attain in hard-to-transfect cells or in vivo. Here we engineer a chemically modified mRNA-encoded adenine base editor that mediates robust editing at various cellular genomic sites together with moderately modified guide RNA, and show its therapeutic potential in correcting pathogenic single nucleotide mutations in cell and animal models of diseases. The optimized chemical modifications of adenine base editor mRNA and guide RNA expand the applicability of CRISPR-associated gene editing tools in vitro and in vivo.

Depletion of TRRAP Induces p53-Independent Senescence in Liver Cancer by Down-Regulating Mitotic Genes.

Hepatocellular carcinoma (HCC) is an aggressive subtype of liver cancer with few effective treatments, and the underlying mechanisms that drive HCC pathogenesis remain poorly characterized. Identifying genes and pathways essential for HCC cell growth will aid the development of new targeted therapies for HCC. Using a kinome CRISPR screen in three human HCC cell lines, we identified transformation/transcription domain-associated protein (TRRAP) as an essential gene for HCC cell proliferation. TRRAP has been implicated in oncogenic transformation, but how it functions in cancer cell proliferation is not established. Here, we show that depletion of TRRAP or its co-factor, histone acetyltransferase KAT5, inhibits HCC cell growth through induction of p53-independent and p21-independent senescence. Integrated cancer genomics analyses using patient data and RNA sequencing identified mitotic genes as key TRRAP/KAT5 targets in HCC, and subsequent cell cycle analyses revealed that TRRAP-depleted and KAT5-depleted cells are arrested at the G2/M phase. Depletion of topoisomerase II alpha (TOP2A), a mitotic gene and TRRAP/KAT5 target, was sufficient to recapitulate the senescent phenotype of TRRAP/KAT5 knockdown. Conclusion: Our results uncover a role for TRRAP/KAT5 in promoting HCC cell proliferation by activating mitotic genes. Targeting the TRRAP/KAT5 complex is a potential therapeutic strategy for HCC.

Adenine base editing in an adult mouse model of tyrosinaemia.

In contrast to traditional CRISPR-Cas9 homology-directed repair, base editing can correct point mutations without supplying a DNA-repair template. Here we show in a mouse model of tyrosinaemia that hydrodynamic tail-vein injection of plasmid DNA encoding the adenine base editor (ABE) and a single-guide RNA (sgRNA) can correct an A>G splice-site mutation. ABE treatment partially restored splicing, generated fumarylacetoacetate hydrolase (FAH)-positive hepatocytes in the liver, and rescued weight loss in mice. We also generated FAH+ hepatocytes in the liver via lipid-nanoparticle-mediated delivery of a chemically modified sgRNA and an mRNA of a codon-optimized base editor that displayed higher base-editing efficiency than the standard ABEs. Our findings suggest that adenine base editing can be used for the correction of genetic diseases in adult animals.

CRISPR-SONIC: targeted somatic oncogene knock-in enables rapid in vivo cancer modeling.

CRISPR/Cas9 has revolutionized cancer mouse models. Although loss-of-function genetics by CRISPR/Cas9 is well-established, generating gain-of-function alleles in somatic cancer models is still challenging because of the low efficiency of gene knock-in. Here we developed CRISPR-based Somatic Oncogene kNock-In for Cancer Modeling (CRISPR-SONIC), a method for rapid in vivo cancer modeling using homology-independent repair to integrate oncogenes at a targeted genomic locus. Using a dual guide RNA strategy, we integrated a plasmid donor in the 3'-UTR of mouse ß-actin, allowing co-expression of reporter genes or oncogenes from the ß-actin promoter. We showed that knock-in of oncogenic Ras and loss of p53 efficiently induced intrahepatic cholangiocarcinoma in mice. Further, our strategy can generate bioluminescent liver cancer to facilitate tumor imaging. This method simplifies in vivo gain-of-function genetics by facilitating targeted integration of oncogenes.

A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing.

CRISPR-Cas9 genome editing has transformed biotechnology and therapeutics. However, in vivo applications of some Cas9s are hindered by large size (limiting delivery by adeno-associated virus [AAV] vectors), off-target editing, or complex protospacer-adjacent motifs (PAMs) that restrict the density of recognition sequences in target DNA. Here, we exploited natural variation in the PAM-interacting domains (PIDs) of closely related Cas9s to identify a compact ortholog from Neisseria meningitidis-Nme2Cas9-that recognizes a simple dinucleotide PAM (N4CC) that provides for high target site density. All-in-one AAV delivery of Nme2Cas9 with a guide RNA targeting Pcsk9 in adult mouse liver produces efficient genome editing and reduced serum cholesterol with exceptionally high specificity. We further expand our single-AAV platform to pre-implanted zygotes for streamlined generation of genome-edited mice. Nme2Cas9 combines all-in-one AAV compatibility, exceptional editing accuracy within cells, and high target site density for in vivo genome editing applications.

Influence of Conventional Surfactants on the Self-Assembly of a Bola Type Amphiphilic Peptide.

Structural and morphological regulation is a distinctly important topic in peptide self-assembly, and is also regarded as the fundamental point in peptide-based biomaterials development. In this paper, we showed that adding anionic surfactant SDS to a bola amphiphilic peptide KI4K could result in the reconstruction of ß-sheet secondary structure besides the changes in self-assembly morphologies from nanotubes to helical ribbons, nanofibers, or straight nanotapes according to the negatively stained transmission electron microscopy, atomic force microscopy, circular dichroism spectroscopy, and Fourier transform infrared spectroscopy results. The inducing effect of SDS was observed at both above and below its CMC but with different transformation rates. Through comparison to other surfactants, including CTAB, C12EO4, and AOT, we proposed that the transitions of KI4K self-assemblies induced by anionic surfactants could be mainly attributed to the effect of hydrophobic interaction and electrostatic attraction between surfactants and peptide molecules. Rheological property measurement and dye adsorption experiments were also carried out to evaluate the properties of hydrogels formed by the peptide/surfactant hybrids. The samples formed self-supporting hydrogels at proper SDS or AOT concentrations, and the charges of hydrogel could be regulated by peptide to surfactant ratio.

Evaluation of the proteasome inhibitor MLN9708 in preclinical models of human cancer.

The proteasome was validated as an oncology target following the clinical success of VELCADE (bortezomib) for injection for the treatment of multiple myeloma and recurring mantle cell lymphoma. Consequently, several groups are pursuing the development of additional small-molecule proteasome inhibitors for both hematologic and solid tumor indications. Here, we describe MLN9708, a selective, orally bioavailable, second-generation proteasome inhibitor that is in phase I clinical development. MLN9708 has a shorter proteasome dissociation half-life and improved pharmacokinetics, pharmacodynamics, and antitumor activity compared with bortezomib. MLN9708 has a larger blood volume distribution at steady state, and analysis of 20S proteasome inhibition and markers of the unfolded protein response confirmed that MLN9708 has greater pharmacodynamic effects in tissues than bortezomib. MLN9708 showed activity in both solid tumor and hematologic preclinical xenograft models, and we found a correlation between greater pharmacodynamic responses and improved antitumor activity. Moreover, antitumor activity was shown via multiple dosing routes, including oral gavage. Taken together, these data support the clinical development of MLN9708 for both hematologic and solid tumor indications.

The relationship among tumor architecture, pharmacokinetics, pharmacodynamics, and efficacy of bortezomib in mouse xenograft models.

Understanding a compound's preclinical pharmacokinetic, pharmacodynamic, and efficacy relationship can greatly facilitate its clinical development. Bortezomib is a first-in-class proteasome inhibitor whose pharmacokinetic/pharmacodynamic parameters are poorly understood in terms of their relationship with efficacy. Here we characterized the bortezomib pharmacokinetic/pharmacodynamic/efficacy relationship in the CWR22 and H460 xenograft models. These studies allowed us to specifically address the question of whether the lack of broad bortezomib activity in solid tumor xenografts was due to insufficient tumor penetration. In vivo studies showed that bortezomib treatment resulted in tumor growth inhibition in CWR22 xenografts, but not in H460 xenografts. Using 20S proteasome inhibition as a pharmacodynamic marker and analyzing bortezomib tumor exposures, we show that efficacy was achieved only when suitable drug exposures drove proteasome inhibition that was sustained over time. This suggested that both the magnitude and duration of proteasome inhibition were important drivers of efficacy. Using dynamic contrast-enhanced magnetic resonance imaging and high-resolution computed tomographic imaging of vascular casts, we characterized the vasculature of CWR22 and H460 xenograft tumors and identified prominent differences in vessel perfusion, permeability, and architecture that ultimately resulted in variations in bortezomib tumor exposure. Comparing and contrasting the differences between a bortezomib-responsive and a bortezomib-resistant model with these techniques allowed us to establish a relationship among tumor perfusion, drug exposure, pharmacodynamic response and efficacy, and provided an explanation for why some solid tumor models do not respond to bortezomib treatment.

Comparison of biochemical and biological effects of ML858 (salinosporamide A) and bortezomib.

Strains within the genus Salinospora have been shown to produce complex natural products having antibiotic and antiproliferative activities. The biochemical basis for the cytotoxic effects of salinosporamide A has been linked to its ability to inhibit the proteasome. Synthetically accessible salinosporamide A (ML858) was used to determine its biochemical and biological activities and to compare its effects with those of bortezomib. ML858 and bortezomib show time- and concentration-dependent inhibition of the proteasome in vitro. However, unlike bortezomib, which is a reversible inhibitor, ML858 covalently binds to the proteasome, resulting in the irreversible inhibition of 20S proteasome activity. ML858 was equipotent to bortezomib in cell-based reporter stabilization assays, but due to intramolecular instability is less potent in long-term assays. ML858 failed to maintain levels of proteasome inhibition necessary to achieve efficacy in tumor models responsive to bortezomib. Our results show that ML858 and bortezomib exhibit different kinetic and pharmacologic profiles and suggest that additional characterization of ML858 is warranted before its therapeutic potential can be fully appreciated.