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Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 5′ overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which are biased toward homonucleotide insertions. Using inverted PAM sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9:gRNA complex influences subsequent NHEJ processing. As with events induced by the site-specific HO endonuclease, CRISPR-Cas9–mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11’s nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of ∼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract, which is currently treated with injected monoclonal antibodies specific for tumor necrosis factor (TNF). We developed and characterized AVX-470, a novel polyclonal antibody specific for human TNF. We evaluated the oral activity of AVX-470m, a surrogate antibody specific for murine TNF, in several well-accepted mouse models of IBD. AVX-470 and AVX-470m were isolated from the colostrum of dairy cows that had been immunized with TNF. The potency, specificity, and affinity of both AVX-470 and AVX-470m were evaluated in vitro and compared with infliximab. AVX-470m was orally administered to mice either before or after induction of colitis, and activity was measured by endoscopy, histopathology, immunohistochemistry, and quantitative measurement of messenger RNA levels. Colitis was induced using either 2,4,6-trinitrobenzene sulfonate or dextran sodium sulfate. AVX-470 and AVX-470m were shown to be functionally comparable in vitro. Moreover, the specificity, neutralizing potency, and affinity of AVX-470 were comparable with infliximab. Orally administered AVX-470m effectively reduced disease severity in several mouse models of IBD. Activity was comparable with that of oral prednisolone or parenteral etanercept. The antibody penetrated the colonic mucosa and inhibited TNF-driven mucosal inflammation with minimal systemic exposure. AVX-470 is a novel polyclonal anti-TNF antibody with an in vitro activity profile comparable to that of infliximab. Oral administration of a surrogate antibody specific for mouse TNF is effective in treating mouse models of IBD, delivering the anti-TNF to the site of inflammation with minimal systemic exposure.

AVX-470 is an oral, bovine-derived, polyclonal antibody designed to target tumor necrosis factor (TNF) locally in the gastrointestinal tract. TNF is central to the dysregulated inflammatory response in IBD. Injected anti-TNF antibodies are highly effective treatments for IBD but are associated with serious side effects from systemic exposure. In a recent double-blind, placebo-controlled first-in-human trial in patients with active ulcerative colitis (UC), AVX-470 administration was associated with dose-dependent increases in clinical and endoscopic remission. Measurement of pro-inflammatory cytokine levels is essential for proof-of-concept and proof-of-mechanism. The aim of this study was to confirm AVX-470 modulation of inflammatory biomarkers in biopsy tissue and serum, and the mechanistic basis of drug effect.MethodsThirty three (33) male and female adults completed 4 weeks of study drug. AVX-470 (0.2 g/day BID, 1.6 g/day BID, and 3.5 g/day TID) and placebo were formulated as enteric-coated capsules for oral administration. Patients underwent colonoscopy with biopsy from 5 colon regions (cecum/ascending, transverse, descending, sigmoid, rectum) at Baseline and after 4 weeks of dosing, and serum samples were collected at defined time points. Endoscopic activity was assessed by centrally-read UCEIS (Ulcerative Colitis Endoscopic Index of Severity) scoring. TNF levels were measured in colon tissue by immunohistochemistry (IHC) as an indicator of drug effects at the target protein. Additional markers including CRP, IL-6, myeloperoxidase (MPO), and TUNEL staining downstream of TNF were analyzed by ELISA and IHC at Baseline and at Week 4.ResultsChanges in markers of inflammation and disease activity were observed in tissue and serum following 4 weeks of AVX-470 treatment. TNF protein levels measured by IHC staining were reduced in colon biopsy tissue from patients in the 3.5g/day dose group at Week 4, confirming direct activity of AVX-470 on the target protein in intestinal tissue. This effect was proportionately greatest in the proximal colon and correlated with both clinical response and the greater reduction of endoscopic inflammatory activity in this bowel segment. Apoptosis in intestinal epithelial cells as measured by TUNEL staining was also reduced in patients from the AVX-470 3.5 g/day dose group at Week 4, particularly in proximal colon. Mean levels of IHC staining for MPO, indicative of neutrophil infiltration, were decreased at Week 4. Levels of serum CRP, a systemic marker of disease, were decreased in the 3.5 g/day treatment group compared to placebo. A dose-dependent reduction in serum IL-6 was also observed with the most pronounced effect in the high dose group compared with placebo.ConclusionsThese findings support proof-of-mechanism for AVX-470 mediated reduction of TNF in intestinal tissue, and the potential for AVX-470 to reduce inflammation, intestinal epithelial cell apoptosis, and disease activity in patients with UC through local drug action in the colon. The overall study design of this trial, which integrates tissue, serum, and stool biomarker analysis with safety, pharmacokinetic and immunogenicity endpoints, represents a new paradigm for streamlined drug development in inflammatory bowel disease.

Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of ∼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response.

The DNA damage checkpoint response is a well-coordinated effort employed by cells when genome integrity has been compromised. Genome integrity is essential to the survival of organisms; therefore, cells have developed numerous ways to sense and resolve DNA damage arising from both endogenous and exogenous sources. In humans, improper repair of DNA lesions or failure to halt cellular division in response to DNA damage can result in the development of cancer and other diseases. Previous work in the DNA damage field has focused on characterization of how cells sense and repair DNA lesions with less emphasis on how DNA damage effects the entire cell. My work contributes to the notion that, in response to DNA damage, cells initiate a well-orchestrated dialogue between the nucleus and the cytoplasm to ensure cell cycle arrest and faithful DNA repair. In this work, I study site-specific double-strand breaks (DSBs) induced by both the HO endonuclease and CRISPR/Cas9 and determine that both require Ku70/80, DNA ligase 4 and Mre11-Rad50-Xrs2 for non-homologous end-joining (NHEJ) repair. We find that CRISPR/Cas9 induced NHEJ insertions are templated as a result of a 1 or 2-nt overhang. Additionally, during the DNA damage checkpoint response I examine the fate of the yeast securin protein, Pds1 and find that it localizes to the vacuole using components of the alkaline phosphatase pathway (ALP) to ensure proper G2/M arrest. I also investigate the requirements for the inositol phosphate kinases during autophagy and find that they are specifically required for genotoxin-induced autophagy (GTA) but not for rapamycin induced autophagy. Collectively, these results highlight the complexity in a cells response to DNA damage and offers insights into therapeutic targets not previously considered in the treatment of cancers.

Development of improved therapies for inflammatory bowel disease (IBD) requires animal models that elucidate underlying mechanisms of disease. Acute radiation syndrome (ARS) has devastating consequences in the gastrointestinal (GI) tract driven in part by cytokine-mediated inflammation that closely resembles the pathophysiology of IBD. Tumor Necrosis Factor (TNF) is central to the dysregulated inflammatory response in IBD, and is strongly implicated in GI ARS. In this study AVX-470 m, a bovine polyclonal neutralizing antibody for murine TNF, is used to characterize the pathogenesis of GI damage in models of GI ARS and IBD.MethodsColitis was induced in C57BL/6 mice by including 3% DSS in drinking water for 5 days. Other C57BL/6 mice were irradiated with 15.9 Gy (∼LD80/30) from a 60Co gamma source, with partial bone marrow shielding. Animals were dosed BID with AVX-470 m (10 mg/day) or saline by oral gavage for up to 14 days. In colon and jejunum tissues, relative mRNA expression levels of cytokines and immune markers was measured by qPCR (Taqman), and protein expression was analyzed by immunohistochemistry (IHC) in paraffin embedded, formalin fixed tissue sections.ResultsBoth mRNA and protein levels of TNF were increased in colon and jejunum tissue, respectively, in the colitis and GI ARS models. IL-1β, IL-6, MMP9, ICAM-1, and TNFR2 mRNA levels were increased in both models, but IL12p40 was increased only in the colitis model, and no change in TNFR1 mRNA levels was detected in either model. Protein expression of MPO was increased in both the colitis and GI ARS models. These data show a remarkable correlation of inflammatory responses between these 2 models of GI injury. In contrast, CD68 and CD3 protein were increased in colon tissue in the colitis model, but neither protein was detected in jejunum in the GI ARS model, suggesting a reduced T-cell component in GI ARS, perhaps due to destruction of these cells by the high levels of ionizing radiation. In both colitis and GI ARS models, treatment with AVX-470 m resulted in a significant decrease in TNF mRNA and protein expression in colon and jejunum tissues, respectively. AVX-470 m also decreased MPO protein expression, and reduced MMP9 mRNA expression, in both models. In the colitis model, IL-6 mRNA levels were decreased by AVX-470 m treatment in colon tissue, while in the GI ARS model IL-6 mRNA levels were transiently increased in jejunum tissue. No change in mRNA levels for ICAM-1 or TNFR1 were seen in either model following AVX-470 m treatment.ConclusionsThese studies show that inflammatory responses in colitis and GI ARS models share a common TNF-mediated neutrophilic component in the colon and jejunum, respectively, but differ with respect to immune cell involvement in response to injury. Further studies will reveal whether these differences reflect variability in timing, tissue localization, or response to the different insults. The fact that AVX-470 m is effective in both models highlights the central role of TNF in inflammation in the GI tract, and supports the therapeutic potential of an oral anti-TNF antibody as a gut-targeted treatment for multiple GI inflammatory diseases.

AVX-470 is a bovine polyclonal anti-TNF antibody that is being developed as an oral therapeutic for the treatment of IBD. The antibody is designed to work locally within the inflamed GI tract, minimizing systemic exposure and the resulting systemic immunosuppression. Oral delivery of a surrogate antibody specific for murine TNF (AVX-470 m) has previously been shown to effectively treat DSS- and TNBS-induced colitis in mice. The goal of this study is to determine the in vitro pharmacological parameters of AVX-470, and to compare some of these parameters to the monoclonal antibody infliximab.MethodsAVX-470 was generated by immunization of pregnant dairy cows with recombinant human TNF. Colostrum was collected and the immunoglobulin fraction was enriched, with a majority of the polyclonal antibody of bovine isotype IgG1. AVX-470 binding to TNF was quantified by direct ELISA on TNF-coated plates. Neutralization of soluble TNF by AVX-470 was determined using the cellbased L929 assay. Sandwich ELISAs were used to determine AVX-470 cross-reactivity with other cytokines. The TNF-specific component of AVX-470 was isolated on a TNF-affinity column and the activity of the affinity-purified and parent materials were compared in both a TNF-specific binding ELISA and in the L929 TNF neutralization assay. Affinity purified AVX-470 (AVX-470A) was used to measure the binding affinity to human TNF by both Surface Plasmon Resonance (SPR) and by competitive ELISA. Flow cytometric analysis was used to assess the ability of AVX-470 to signal through transmembrane TNF and induce apoptosis in PMA and Ionomycin activated human PBMC, as measured by annexin V and propidium iodide uptake.ResultsAVX-470 bound to human TNF in a solid phase ELISA with a high titer, and robustly neutralized soluble human TNF in a cell-based assay. While AVX-470 bound strongly to human TNF, it bound less well to non-human primate TNF, and bound weakly to other species (human>cynomolgus monkey/rhesus macaque>dog>>rodent and cow). AVX-470 demonstrated no cross reactivity to TNFrelated cytokines. By both SPR and competitive ELISA, AVX-470A showed an affinity to TNF in the high picomolar range, comparable to that of infliximab. The monoclonal antibody infliximab displayed a very sharp inhibition curve while the polyclonal AVX-470A displayed a shallower inhibition curve, indicative of a broad range of antibodies directed against multiple epitopes on TNF. AVX-470 induced apoptosis by reverse signaling in activated human PBMC, similar to that of infliximab, while control bovine immunoglobulin showed no effect on apoptosis.Conclusion(s)AVX-470 is a potent, specific, high affinity anti-TNF antibody which binds to TNF in a solid phase binding assay, neutralizes soluble TNF in the L929 cell-based assay, and induces reverse signaling through membrane TNF as measured by induction of apoptosis. In affinity measurements, TNF neutralization, and apoptosis studies, AVX-470 was comparable to infliximab. AVX-470 has some reactivity to non-human primate TNF, but little to no reactivity to other species tested. This finding has enabled a toxicology study in cynomolgus monkeys. Taken together, these in vitro data support the use of AVX-470 as a therapeutic agent for the treatment of IBD.