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Traditional methods for chimeric antigen receptor (CAR) T manufacturing utilize viral vectors, ex vivo activation and expansion of T cells to achieve clinically relevant cell numbers, which leads to an exhausted T cell phenotype, high manufacturing costs, and treatment delays. The UltraCAR-T platform is designed to overcome these limitations using our advanced non-viral gene delivery system and a rapid, overnight manufacturing process (Blood 2019 134 (Supplement_1):2660; Blood 2020 136 (Supplement 1):17); Cancer Research 2020 80 (16Suppl):6593). UltraCAR-T cells, which express antigen specific CAR, membrane-bound IL-15 (mbIL15), and kill switch genes, are manufactured at the medical center's cGMP facility using autologous T cells and administered back to the patient only one day after gene transfer. UltraCAR-T cells are currently under clinical investigation for hematological (NCT03927261) and solid tumors (NCT03907527). Here we describe the advancement of the UltraCAR-T platform to address the inhibitory tumor microenvironment by incorporating intrinsic checkpoint blockade without the need for complex and expensive gene editing techniques. PRGN-3007, based on the next generation of the UltraCAR-T platform, is engineered to simultaneously express CAR for targeting receptor tyrosine kinase-like orphan receptor 1 (ROR1), which is overexpressed on many hematological and solid tumors; mbIL15 for enhanced in vivo expansion and persistence; kill switch for improved safety profile; and a novel mechanism for the intrinsic blockade of PD-1 gene expression. This approach of intrinsic blockade of PD-1 expression, only on UltraCAR-T cells, is aimed to avoid systemic toxicity and high cost of checkpoint inhibitors by eliminating the need for combination treatment. PRGN-3007 is manufactured using the already established rapid and streamlined UltraCAR-T manufacturing process. PRGN-3007 was generated using multiple healthy donor T cells using multi-cistronic non-viral vector and the overnight manufacturing process. The co-expression of CAR, mbIL15 and kill switch transgenes was confirmed by flow cytometry, western blotting, and qPCR. Furthermore, PRGN-3007 showed significant reduction in PD-1 expression on UltraCAR-T cells compared to ROR1 CAR-T cells lacking PD-1 blockade (Control ROR1 CAR-T). The downregulation of PD-1 expression on PRGN-3007 resulted in enhanced ROR1-specific cytotoxicity and release of inflammatory cytokines upon co-culture with various ROR1 + PD-L1 + hematological and solid tumor cells compared to Control ROR1 CAR-T, especially at low effector to target cell ratios. Single-cell cytokine proteomics showed that the downregulation of PD-1 expression on PRGN-3007 resulted in a significantly higher number of polyfunctional CAR-T cells compared to Control ROR1 CAR-T. Expression of mbIL15 on UltraCAR-T, with or without downregulation of PD-1 expression, resulted in robust expansion in presence of ROR1 antigen, lack of autonomous expansion in absence of ROR1, and durable persistence even in absence of exogenous cytokines in vitro. Furthermore, PRGN-3007 was selectively and effectively eliminated by the kill switch activator antibody treatment. A single administration of PRGN-3007, only one day after gene transfer, effectively reduced tumor burden and significantly improved overall survival (p In summary, these preclinical data highlight the overall safety and improved efficacy of incorporating intrinsic downregulation of PD-1 expression on UltraCAR-T cells using non-viral gene delivery and the established rapid, decentralized manufacturing process. These data provide a strong rationale for the evaluation of PRGN-3007 for the treatment of ROR1 + malignancies. Figure: Overall survival in in an established model of mantle cell lymphoma in NSG mice. Tumor cells were engrafted in mice on Day 0 and treatments were administered on Day 8. Data shown is from 8 mice/group at the start of the study. * p Figure 1 Figure 1. Disclosures Chan: Precigen, Inc: Current Employment, Current equity holder in publicly-traded company. Scott: Precigen, Inc: Current Employment. Du: Precigen, Inc: Current Employment. Bolinger: Precigen, Inc: Current Employment. Poortman: Precigen, Inc: Current Employment. Shepard: Precigen, Inc: Current Employment. Koenitzer: Precigen, Inc: Current Employment. Govekung: Precigen, Inc: Current Employment. Sailor: Precigen, Inc: Current Employment. Johnson: Precigen, Inc: Current Employment. Plummer: Precigen, Inc: Current Employment. Zilko: Precigen, Inc: Current Employment. Dutta: Precigen, Inc: Current Employment. Kunchithapautham: Precigen, Inc: Current Employment. Athwal: Precigen, Inc: Current Employment. Klocke: Precigen, Inc: Current Employment. Zinser: Precigen, Inc: Current Employment. Abdeladhim: Precigen, Inc: Current Employment. Ahmad: Precigen, Inc: Current Employment; Kite, A Gilead Company: Ended employment in the past 24 months. Metenou: Precigen, Inc: Current Employment. Semnani: Precigen, Inc: Current Employment. Brough: Precigen, Inc: Current Employment, Current equity holder in publicly-traded company. Shah: Precigen: Current Employment, Current equity holder in publicly-traded company. Sabzevari: Precigen: Current Employment, Current equity holder in publicly-traded company; Kinnate BioPharma: Membership on an entity's Board of Directors or advisory committees; Compass Therapeutics: Current equity holder in publicly-traded company.

To elucidate the role of hepatic cytochrome P450 oxidoreductase (POR) in lipid metabolism, we characterized perturbations in lipid homeostasis in a mouse model deficient in liver POR. Using an integrative approach in which transcriptomics, lipidomics, and various bioinformatic algorithms were employed, a disruption in liver lipid mobilization, oxidation, and electron transport functions were identified. Analyzing the promoters of genes in these biological processes identified common binding motifs for nuclear receptors sensitive to lipid status, while Srebp-1c binding sites were only identified in genes involved in lipid metabolism. POR-null mice had drastic increases in hepatic lipid content (diacylglycerols, triacylglycerols, phosphatidylcholine, and cholesterol esters) and a specific enrichment in n-7 and n-9 monounsaturated fatty acids (FAs). It was found that while transporters involved in peroxisomal FA oxidation were induced, mitochondrial oxidation appeared to be more tightly controlled, supporting the increase in monounsaturated FAs. Genes coding for hepatic transporters were differentially expressed, where lipid uptake was induced and efflux repressed, indicating that in the absence of hepatic POR the liver serves as a lipid reservoir. Furthermore, while significant changes in intestinal gene expression were found in POR-deficient mice, only minor changes to plasma and intestinal lipid content were observed. Thus, while liver POR plays an important role regulating gene expression and lipid metabolism locally, the hepatic deficiency of this enzyme reverberates throughout the biological system and produces a coordinated response to the low levels of circulating cholesterol and bile.

Zn finger proteins (ZFPs) of the C2/H2 type in Xenopus laevis are encoded by a multigene family comprising several hundred members. Based upon conserved sequence features outside the Zn finger region, ZFPs can be subdivided into distinct subfamilies. Two of such subfamilies are characterized by conserved, N-terminal amino acid sequences termed the FAX and the FAR Domain. Here we present data suggesting that the zinc finger proteins of the FAR-ZFP subfamily are targets for CK II mediated phosphorylation. Expression of these proteins during oogenesis coincides with CK II activity in unfertilized eggs. Additionally, we have found that XlcOF 7.1, a member of the FAX-ZFP subfamily, is also phosphorylated by CK II. The target sites for in vitro phosphorylation are localized within the conserved N-terminal domains but not within the Zn finger regions. However, amino acid sequence comparison revealed that individual phosphoacceptor sites are not generally conserved among all members of the respective ZFP subfamilies. The relevance of a potential CK II phosphorylation for the regulation of ZFP activity in vivo is discussed.

The function of the aryl hydrocarbon receptor (AhR) in mediating the biological effect to environmental pollutants is well established. However, accumulated evidence indicates a wide range of physiological and pathological functions mediated by the AhR, suggesting the existence of endogenous AhR ligand(s). The nature of an AhR ligand remain elusive; however, it is known that the AhR is activated by several compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin or the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole. In this study, we show that physiological concentrations of tryptamine (TA) lead to induction of cytochrome P4501A1 transcription through an AhR-dependent mechanism. In addition, we show that activation of the AhR by TA requires a functional monoamino oxidase system, suggesting that TA acts as an AhR proligand possibly by converting to a high-affinity AhR ligand. Taken together, we show a possible mechanism, through which AhR signaling is activated by endogenous conversion of TA involving monoamine oxidases.

A stringent control of homeostasis is critical for functional maintenance and survival of neurons. In the mammalian retina, the basic motif leucine zipper transcription factor NRL determines rod versus cone photoreceptor cell fate and activates the expression of many rod-specific genes. Here, we report an integrated analysis of NRL-centered gene regulatory network by coupling chromatin immunoprecipitation followed by high-throughput sequencing (ChIP–Seq) data from Illumina and ABI platforms with global expression profiling and in vivo knockdown studies. We identified approximately 300 direct NRL target genes. Of these, 22 NRL targets are associated with human retinal dystrophies, whereas 95 mapped to regions of as yet uncloned retinal disease loci. In silico analysis of NRL ChIP–Seq peak sequences revealed an enrichment of distinct sets of transcription factor binding sites. Specifically, we discovered that genes involved in photoreceptor function include binding sites for both NRL and homeodomain protein CRX. Evaluation of 26 ChIP–Seq regions validated their enhancer functions in reporter assays. In vivo knockdown of 16 NRL target genes resulted in death or abnormal morphology of rod photoreceptors, suggesting their importance in maintaining retinal function. We also identified histone demethylase Kdm5b as a novel secondary node in NRL transcriptional hierarchy. Exon array analysis of flow-sorted photoreceptors in which Kdm5b was knocked down by shRNA indicated its role in regulating rod-expressed genes. Our studies identify candidate genes for retinal dystrophies, define cis-regulatory module(s) for photoreceptor-expressed genes and provide a framework for decoding transcriptional regulatory networks that dictate rod homeostasis., Author Summary The rod and cone photoreceptors in the retina are highly specialized neurons that capture photons under dim and bright light, respectively. Loss of rod photoreceptors is an early clinical manifestation in most retinal neurodegenerative diseases that eventually result in cone cell death and blindness. The transcription factor NRL is a key regulator of rod photoreceptor cell fate and gene expression. Here, we report an integrated analysis of the global transcriptional targets of NRL. We have discovered that both NRL and CRX binding sites are present in genes involved in photoreceptor function, implying their close synergistic relationship. In vivo loss-of-function analysis of 16 NRL target genes in the mouse retina resulted in death or abnormal morphology of photoreceptor cells. Furthermore, we identified histone demethylase Kdm5b as a secondary node in the NRL-centered gene regulatory network. Our studies identify NRL target genes as excellent candidates for mutation screening of patients with retinal degenerative diseases, and they provide the foundation for elucidating regulation of rod homeostasis and targets for therapeutic intervention in diseases involving photoreceptor dysfunction.

The zinc finger motif defines a large superfamily of nucleic acid binding proteins. Conserved amino acid sequence elements associated with structurally variant zinc finger clusters define subfamilies of zinc finger proteins (ZFPs). The FAR domain (Finger Associated Repeats) is a novel type of repeat element found at the amino-terminus in a subfamily of Xenopus laevis ZFPs. Northern blot analyses of three different members of the FAR subfamily (XFO 6, XFO 9-3 and XFG 68) revealed that each of these genes is transcribed during oogenesis, embryogenesis and in all investigated tissues of adult animals thereby indicating a ubiquitous distribution of transcripts. All FAR-ZFPs tested so far have specific RNA homopolymer binding activity; they associate preferentially with poly(U). The FAR repeats possess limited primary sequence homology with a sequence in the nucleolar shuttling protein NO38, within a region that contains a casein kinase II phosphorylation site.

Motivation: Promoter analysis is an essential step on the way to identify regulatory networks. A prerequisite for successful promoter analysis is the prediction of potential transcription factor binding sites (TFBS) with reasonable accuracy. The next steps in promoter analysis can be tackled only with reliable predictions, e.g. finding phylogenetically conserved patterns or identifying higher order combinations of sites in promoters of co-regulated genes. Results: We present a new version of the program MatInspector that identifies TFBS in nucleotide sequences using a large library of weight matrices. By introducing a matrix family concept, optimized thresholds, and comparative analysis, the enhanced program produces concise results avoiding redundant and false-positive matches. We describe a number of programs based on MatInspector allowing in-depth promoter analysis (DiAlignTF, FrameWorker) and targeted design of regulatory sequences (SequenceShaper). Availability: MatInspector and the other programs described here can be used online at http://www.genomatix.de/matinspector.html. Access is free after registration within certain limitations (e.g. the number of analysis per month is currently limited to 20 analyses of arbitrary sequences). Contact: cartharius@genomatix.de Supplementary information: http://www.genomatix.de/matinspector.html