A precise and efficient adenine base editor.

Adenine base editors (ABEs) are novel genome-editing tools, and their activity has been greatly enhanced by eight additional mutations, thus named ABE8e. However, elevated catalytic activity was concomitant with frequent generation of bystander mutations. This bystander effect precludes its safe applications required in human gene therapy. To develop next-generation ABEs that are both catalytically efficient and positionally precise, we performed combinatorial engineering of NG-ABE8e. We identify a novel variant (NG-ABE9e), which harbors nine mutations. NG-ABE9e exhibits robust and precise base-editing activity in human cells, with more than 7-fold bystander editing reduction at some sites, compared with NG-ABE8e. To demonstrate its practical utility, we used NG-ABE9e to correct the frequent T17M mutation in Rhodopsin for autosomal dominant retinitis pigmentosa. It reduces bystander editing by ∼4-fold while maintaining comparable efficiency. NG-ABE9e possesses substantially higher activity than NG-ABEmax and significantly lower bystander editing than NG-ABE8e in rice. Therefore, this study provides a versatile and improved adenine base editor for genome editing.

Small-molecule compounds boost genome-editing efficiency of cytosine base editor.

Cytosine base editor (CBE) enables targeted C-to-T conversions at single base-pair resolution and thus has potential therapeutic applications in humans. However, the low efficiency of the system limits practical use of this approach. We reported a high-throughput human cells-based reporter system that can be harnessed for quickly measuring editing activity of CBE. Screening of 1813 small-molecule compounds resulted in the identification of Ricolinostat (an HDAC6 inhibitor) that can enhance the efficiency of BE3 in human cells (2.45- to 9.21-fold improvement). Nexturastat A, another HDAC6 inhibitor, could also increase BE3-mediated gene editing by 2.18- to 9.95-fold. Ricolinostat and Nexturastat A also boost base editing activity of the other CBE variants (BE4max, YE1-BE4max, evoAPOBEC1-BE4max and SpRY-CBE4max, up to 8.32-fold). Meanwhile, combined application of BE3 and Ricolinostat led to >3-fold higher efficiency of correcting a pathogenic mutation in ABCA4 gene related to Stargardt disease in human cells. Moreover, we demonstrated that our strategy could be applied for efficient generation of mouse models through direct zygote injection and base editing in primary human T cells. Our study provides a new strategy to improve the activity and specificity of CBE in human cells. Ricolinostat and Nexturastat A augment the effectiveness and applicability of CBE.

Spatially resolved transcriptome of the aging mouse brain.

Brain aging is associated with cognitive decline, memory loss and many neurodegenerative disorders. The mammalian brain has distinct structural regions that perform specific functions. However, our understanding in gene expression and cell types within the context of the spatial organization of the mammalian aging brain is limited. Here we generated spatial transcriptomic maps of young and old mouse brains. We identified 27 distinguished brain spatial domains, including layer-specific subregions that are difficult to dissect individually. We comprehensively characterized spatial-specific changes in gene expression in the aging brain, particularly for isocortex, the hippocampal formation, brainstem and fiber tracts, and validated some gene expression differences by qPCR and immunohistochemistry. We identified aging-related genes and pathways that vary in a coordinated manner across spatial regions and parsed the spatial features of aging-related signals, providing important clues to understand genes with specific functions in different brain regions during aging. Combined with single-cell transcriptomics data, we characterized the spatial distribution of brain cell types. The proportion of immature neurons decreased in the DG region with aging, indicating that the formation of new neurons is blocked. Finally, we detected changes in information interactions between regions and found specific pathways were deregulated with aging, including classic signaling WNT and layer-specific signaling COLLAGEN. In summary, we established a spatial molecular atlas of the aging mouse brain (http://sysbio.gzzoc.com/Mouse-Brain-Aging/), which provides important resources and novel insights into the molecular mechanism of brain aging.

Rational Selection of CRISPR-Cas Triggering Homology-Directed Repair in Human Cells.

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated) nucleases have been widely applied for genome engineering. Cas9 (Streptococcus pyogenes Cas9 [SpCas9] and Staphylococcus aureus Cas9 [SaCas9]) and Cpf1 (i.e., Francisella novicida U112 Cpf1 [FnCpf1], also named FnCas12a) were harnessed to perform gene editing in human cells. Precise genetic modification by homology-directed repair (HDR) is an attractive approach for in situ gene correction. However, so far, the comparative efficiencies of HDR mediated by different CRISPR orthologs remain unknown. To address this question, in this study, we developed a reporter system to investigate HDR efficiencies triggered by various CRISPR orthologs. We found that SpCas9 and SaCas9, the two most commonly used Cas9 enzymes, possessed a similar ability to induce HDR. Interestingly, with the increasing amount of coding plasmids or additional nuclear localization sequences, FnCpf1 could improve the HDR efficacy. Collectively, our study provides insights for the rational selection of appropriate tools for human genome manipulation.

Human cell based directed evolution of adenine base editors with improved efficiency.

Adenine base editors (ABE) are genome-editing tools that have been harnessed to introduce precise A•T to G•C conversion. However, the low activity of ABE at certain sites remains a major bottleneck that precludes efficacious applications. Here, to address it, we develop a directional screening system in human cells to evolve the deaminase component of the ABE, and identify three high-activity NG-ABEmax variants: NG-ABEmax-SGK (R101S/D139G/E140K), NG-ABEmax-R (Q154R) and NG-ABEmax-K (N127K). With further engineering, we create a consolidated variant [NG-ABEmax-KR (N127K/Q154R)] which exhibit superior editing activity both in human cells and in mouse disease models, compared to the original NG-ABEmax. We also find that NG-ABEmax-KR efficiently introduce natural mutations in gamma globin gene promoters with more than four-fold increase in editing activity. This work provides a broadly applicable, rapidly deployable platform to directionally screen and evolve user-specified traits in base editors that extend beyond augmented editing activity.

Development of a Simple and Quick Method to Assess Base Editing in Human Cells.

Base editing is a form of genome editing that can directly convert a single base (C or A) to another base (T or G), which is of great potential in biomedical applications. The broad application of base editing is limited by its low activity and specificity, which still needs to be resolved. To address this, a simple and quick method for the determination of its activity/specificity is highly desired. Here, we developed a novel system, which could be harnessed for quick detection of editing activity and specificity of base editors (BEs) in human cells. Specifically, multiple cloning sites (MCS) were inserted into the human genome via lentivirus, and base editing targeting the MCS was performed with BEs. The base editing activities were assessed by specific restriction enzymes. The whole process only includes nucleotide-based targeting the MCS, editing, PCR, and digestion, thus, we named it NOTEPAD. This straightforward approach could be easily accessed by molecular biology laboratories. With this method, we could easily determine the BEs editing efficiency and pattern. The results revealed that BEs triggered more off-target effects in the genome than on plasmids including genomic indels (insertions and deletions). We found that ABEs (adenine base editors) had better fidelity than CBEs (cytosine base editors). Our system could be harnessed as a base editing assessment platform, which would pave the way for the development of next-generation BEs.

HIV protease inhibitors enhance the efficacy of irradiation.

Tumor vascular endothelium is rather resistant to the cytotoxic effects of radiation. The HIV protease inhibitors (HPI) amprenavir, nelfinavir, and saquinavir have previously been shown to sensitize tumor cells to the cytotoxic effects of radiation. Additionally, this class of drug has been shown to inhibit angiogenesis and tumor cell migration. Therefore, in the current study, we wanted to determine whether HPIs could enhance the effect of radiation on endothelial function. Our study shows that HPIs, particularly nelfinavir, significantly enhance radiations effect on human umbilical vein endothelial cells (HUVEC) and tumor vascular endothelium. We show that pretreatment of HUVEC with nelfinavir results in enhanced cytotoxicity, including increased apoptosis, when combined with radiation. Moreover, using several functional assays, we show that combination treatment effectively blocks endothelial cell migration and organization. These findings were accompanied by attenuation of Akt phosphorylation, a known pathway for radioresistance. Last, in vivo analysis of tumor microvasculature destruction showed a more than additive effect for nelfinavir and radiation. This study shows that HPIs can enhance the effect of ionizing radiation on vascular endothelium. Therefore, the Food and Drug Administration-approved drug, nelfinavir, may be an effective radiosensitizer in the clinic.

The laminin binding α3 and α6 integrins cooperate to promote epithelial cell adhesion and growth.

Integrins, the major receptors for cell-extracellular matrix (ECM) interactions, regulate multiple cell biological processes including adhesion, migration, proliferation and growth factor-dependent signaling. The principal laminin (LM) binding integrins α3ß1, α6ß1 and α6ß4 are usually co-expressed in cells and bind to multiple laminins with different affinities making it difficult to define their specific function. In this study, we generated kidney epithelial collecting duct (CD) cells that lack both the α3 and α6 integrin subunits. This deletion impaired cell adhesion and migration to LM-332 and LM-511 more than deleting α3 or α6 alone. Cell adhesion mediated by both α3ß1 and α6 integrins was PI3K independent, but required K63-linked polyubiquitination of Akt by the ubiquitin-modifying enzyme TRAF6. Moreover, we provide evidence that glial-derived neurotrophic factor (GDNF) and fibroblast growth factor 10 (FGF10)- mediated cell signaling, spreading and proliferation were severely compromised in double integrin α3/α6- but not single α3- or α6-null CD cells. Interestingly, these growth factor-dependent cell functions required both PI3K- and TRAF6-dependent Akt activation. These data suggest that expression of the integrin α3 or α6 subunit is sufficient to mediate GDNF- and FGF10-dependent spreading, proliferation and signaling on LM-511. Thus, our study shows that α3 and α6 containing integrins promote distinct functions and signaling by CD cells on laminin substrata.

Histone deacetylase (HDAC) inhibitor LBH589 increases duration of gamma-H2AX foci and confines HDAC4 to the cytoplasm in irradiated non-small cell lung cancer.

Histone deacetylases (HDAC) have been identified as therapeutic targets due to their regulatory function in DNA structure and organization. LBH589 is a novel inhibitor of class I and II HDACs. We studied the effect of LBH589 and ionizing radiation (IR) on DNA repair in two human non-small cell lung cancer (NSCLC) cell lines (H23 and H460). gamma-H2AX foci present at DNA double-strand breaks (DSBs) were detected in the nuclei following 3 Gy irradiation for up to 6 hours. LBH589 administered before irradiation increased the duration of gamma-H2AX foci beyond 24 hours. Furthermore, radiation alone induced translocation of HDAC4 to the nucleus. In contrast, treatment with LBH589 followed by irradiation resulted in HDAC4 confinement to the cytoplasm, indicating that HDAC inhibition affects the nuclear localization of HDAC4. The findings that LBH589 confines HDAC4 to the cytoplasm and increases the duration of gamma-H2AX foci in irradiated cell lines suggest that HDAC4 participates in DNA damage signaling following IR. Annexin-propidium iodide flow cytometry assays, cell morphology studies, and cleaved caspase-3 Western blot analysis revealed a synergistic effect of LBH589 with IR in inducing apoptosis. Clonogenic survival showed a greater than additive effect when LBH589 was administered before irradiation compared with irradiation alone. In vivo tumor volume studies showed a growth delay of 20 days with combined treatment compared with 4 and 2 days for radiation or LBH589 alone. This study identifies HDAC4 as a biomarker of LBH589 activity and recognizes the ability of LBH589 to sensitize human NSCLC to radiation-induced DNA DSBs.

Integrin alpha6 maintains the structural integrity of the kidney collecting system.

Laminins are a major constituent of the basement membranes of the kidney collecting system. Integrins, transmembrane receptors formed by non-covalently bound α and ß subunits, serve as laminin receptors, but their role in development and homeostasis of the kidney collecting system is poorly defined. Integrin α3ß1, one of the major laminin receptors, plays a minor role in kidney collecting system development, while the role of α6 containing integrins (α6ß1 and α6ß4), the other major laminin receptors, is unknown. Patients with mutations in α6 containing integrins not only develop epidermolysis bullosa, but also have abnormalities in the kidney collecting system. In this study, we show that selectively deleting the α6 or ß4 integrin subunits at the initiation of ureteric bud development in mice does not affect morphogenesis. However, the collecting system becomes dilated and dysmorphic as the mice age. The collecting system in both null genotypes was also highly susceptible to unilateral ureteric obstruction injury with evidence of excessive tubule dilatation and epithelial cell apoptosis. Mechanistically, integrin α6-null collecting duct cells are unable to withstand high mechanical force when adhered to laminin. Thus, we conclude that α6 integrins are important for maintaining the integrity of the kidney collecting system by enhancing tight adhesion of the epithelial cells to the basement membrane. These data give a mechanistic explanation for the association between kidney collecting system abnormalities in patients and epidermolysis bullosa.

Integrin α3ß1 regulates kidney collecting duct development via TRAF6-dependent K63-linked polyubiquitination of Akt.

The collecting system of the kidney develops from the ureteric bud (UB), which undergoes branching morphogenesis, a process regulated by multiple factors, including integrin-extracellular matrix interactions. The laminin (LM)-binding integrin α3ß1 is crucial for this developmental program; however, the LM types and LM/integrin α3ß1-dependent signaling pathways are poorly defined. We show that α3 chain-containing LMs promote normal UB branching morphogenesis and that LM-332 is a better substrate than LM-511 for stimulating integrin α3ß1-dependent collecting duct cell functions. We demonstrate that integrin α3ß1-mediated cell adhesion to LM-332 modulates Akt activation in the developing collecting system and that Akt activation is PI3K independent but requires decreased PTEN activity and K63-linked polyubiquitination. We identified the ubiquitin-modifying enzyme TRAF6 as an interactor with the integrin ß1 subunit and regulator of integrin α3ß1-dependent Akt activation. Finally, we established that the developmental defects of TRAF6- and integrin α3-null mouse kidneys are similar. Thus K63-linked polyubiquitination plays a previously unrecognized role in integrin α3ß1-dependent cell signaling required for UB development and may represent a novel mechanism whereby integrins regulate signaling pathways.

Kinomic profiling approach identifies Trk as a novel radiation modulator.

BACKGROUND: Ionizing radiation treatment is used in over half of all cancer patients, thus determining the mechanisms of response or resistance is critical for the development of novel treatment approaches. MATERIALS AND METHODS: In this report, we utilize a high-content peptide array platform that performs multiplex kinase assays with real-time kinetic readout to investigate the mechanism of radiation response in vascular endothelial cells. We applied this technology to irradiated human umbilical vein endothelial cells (HUVEC). RESULTS: We identified 49 specific tyrosine phosphopeptides that were differentially affected by irradiation over a time course of 1h. In one example, the Tropomyosin receptor kinase (Trk) family members, TrkA and TrkB, showed transient activation between 2 and 15 min following irradiation. When we targeted TrkA and TrkB using small molecule inhibitors, HUVEC were protected from radiation damage. Conversely, stimulation of TrkA using gambogic amide promoted radiation enhancement. CONCLUSIONS: Thus, we show that our approach not only can identify rapid changes in kinase activity but also identify novel targets such as TrkA. TrkA inhibition resulted in radioprotection that correlated with enhanced repair of radiation-induced damage while TrkA stimulation by gambogic amide produced radiation sensitization.

Enzastaurin (LY317615), a protein kinase C beta selective inhibitor, enhances antiangiogenic effect of radiation.

PURPOSE: Angiogenesis has generated interest in oncology because of its important role in cancer growth and progression, particularly when combined with cytotoxic therapies, such as radiotherapy. Among the numerous pathways influencing vascular growth and stability, inhibition of protein kinase B(Akt) or protein kinase C(PKC) can influence tumor blood vessels within tumor microvasculature. Therefore, we wanted to determine whether PKC inhibition could sensitize lung tumors to radiation. METHODS AND MATERIALS: The combination of the selective PKCbeta inhibitor Enzastaurin (ENZ, LY317615) and ionizing radiation were used in cell culture and a mouse model of lung cancer. Lung cancer cell lines and human umbilical vascular endothelial cells (HUVEC) were examined using immunoblotting, cytotoxic assays including cell proliferation and clonogenic assays, and Matrigel endothelial tubule formation. In vivo, H460 lung cancer xenografts were examined for tumor vasculature and proliferation using immunohistochemistry. RESULTS: ENZ effectively radiosensitizes HUVEC within in vitro models. Furthermore, concurrent ENZ treatment of lung cancer xenografts enhanced radiation-induced destruction of tumor vasculature and proliferation by IHC. However, tumor growth delay was not enhanced with combination treatment compared with either treatment alone. Analysis of downstream effectors revealed that HUVEC and the lung cancer cell lines differed in their response to ENZ and radiation such that only HUVEC demonstrate phosphorylated S6 suppression, which is downstream of mTOR. When ENZ was combined with the mTOR inhibitor, rapamycin, in H460 lung cancer cells, radiosensitization was observed. CONCLUSION: PKC appears to be crucial for angiogenesis, and its inhibition by ENZ has potential to enhance radiotherapy in vivo.

Bone marrow X kinase-mediated signal transduction in irradiated vascular endothelium.

Radiation-induced activation of the phosphatidyl inositol-3 kinase/Akt signal transduction pathway requires Akt binding to phosphatidyl-inositol phosphates (PIP) on the cell membrane. The tyrosine kinase bone marrow X kinase (Bmx) binds to membrane-associated PIPs in a manner similar to Akt. Because Bmx is involved in cell growth and survival pathways, it could contribute to the radiation response within the vascular endothelium. We therefore studied Bmx signaling within the vascular endothelium. Bmx was activated rapidly in response to clinically relevant doses of ionizing radiation. Bmx inhibition enhanced the efficacy of radiotherapy in endothelial cells as well as tumor vascular endothelium in lung cancer tumors in mice. Retroviral shRNA knockdown of Bmx protein enhanced human umbilical vascular endothelial cell (HUVEC) radiosensitization. Furthermore, pretreatment of HUVEC with a pharmacologic inhibitor of Bmx, LFM-A13, produced significant radiosensitization of endothelial cells as measured by clonogenic survival analysis and apoptosis as well as functional assays including cell migration and tubule formation. In vivo, LFM-A13, when combined with radiation, resulted in significant tumor microvascular destruction as well as enhanced tumor growth delay. Bmx therefore represents a molecular target for the development of novel radiosensitizing agents.