Review of Safety Data for Adenovirus 5 as a Delivery Vector for Intratumoral Cancer Gene Therapy.

With efficient transduction across most cell types and larger packaging capacity, Adenovirus 5 (Ad5) makes an attractive choice as a viral vector. However, a reported past mortality and known immunogenicity cast doubt on the safety of its use. An online database search was performed for all clinical trials administering intratumoral injection of gene therapy packaged in Ad5, being conducted in the United States, and using the Common Terminology Criteria for Adverse Events (CTCAE).

Exon skipping induced by CRISPR-directed gene editing regulates the response to chemotherapy in non-small cell lung carcinoma cells.

We have been developing CRISPR-directed gene editing as an augmentative therapy for the treatment of non-small cell lung carcinoma (NSCLC) by genetic disruption of Nuclear Factor Erythroid 2-Related Factor 2 (NRF2). NRF2 promotes tumor cell survival in response to therapeutic intervention and thus its disablement should restore or enhance effective drug action. Here, we report how NRF2 disruption leads to collateral damage in the form of CRISPR-mediated exon skipping.

Deconvolution of Complex DNA Repair (DECODR): Establishing a Novel Deconvolution Algorithm for Comprehensive Analysis of CRISPR-Edited Sanger Sequencing Data.

During CRISPR-directed gene editing, multiple gene repair mechanisms interact to produce a wide and largely unpredictable variety of sequence changes across an edited population of cells. Shortcomings inherent to previously available proposal-based insertion and deletion (indel) analysis software necessitated the development of a more comprehensive tool that could detect a larger range and variety of indels while maintaining the ease of use of tools currently available. To that end, we developed convolution of mplex NA epair (DECODR).

Kinetics of Nuclear Uptake and Site-Specific DNA Cleavage during CRISPR-Directed Gene Editing in Solid Tumor Cells.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-directed gene editing is approaching clinical implementation in cancer. Thus, it is imperative to define the molecular framework upon which safe and efficacious therapeutic strategies can be built. Two important reaction parameters include the biological time frame within which the CRISPR/Cas complex enters the nucleus and executes gene editing, and the method of discrimination that the CRISPR/Cas complex utilizes to target tumor cell, but not normal cell, genomes.

Auxin-dependent control of a plasmodesmal regulator creates a negative feedback loop modulating lateral root emergence.

Lateral roots originate from initial cells deep within the main root and must emerge through several overlying layers. Lateral root emergence requires the outgrowth of the new primordium (LRP) to coincide with the timely separation of overlying root cells, a developmental program coordinated by the hormone auxin. Here, we report that in Arabidopsis thaliana roots, auxin controls the spatiotemporal expression of the plasmodesmal regulator PDLP5 in cells overlying LRP, creating a negative feedback loop.

Cas9/gRNA-mediated genome editing of yeast mitochondria and chloroplasts.

We present a new approach to edit both mitochondrial and chloroplast genomes. Organelles have been considered off-limits to CRISPR due to their impermeability to most RNA and DNA. This has prevented applications of Cas9/gRNA-mediated genome editing in organelles while the tool has been widely used for engineering of nuclear DNA in a number of organisms in the last several years.

Arabidopsis casein kinase 1-like 6 contains a microtubule-binding domain and affects the organization of cortical microtubules,

Members of the casein kinase 1 (CK1) family are evolutionarily conserved eukaryotic protein kinases that are involved in various cellular, physiological, and developmental processes in yeast and metazoans, but the biological roles of CK1 members in plants are not well understood. Here, we report that an Arabidopsis (Arabidopsis thaliana) CK1 member named casein kinase 1-like 6 (CKL6) associates with cortical microtubules in vivo and phosphorylates tubulins in vitro. The unique C-terminal domain of CKL6 was shown to contain the signal that allows localization of CKL6 to the cortical microtubules.

Calcium-regulated phosphorylation of soybean serine acetyltransferase in response to oxidative stress.

Glycine max serine acetyltransferase 2;1 (GmSerat2;1) is a member of a family of enzymes that catalyze the first reaction in the biosynthesis of cysteine from serine. It was identified by interaction cloning as a protein that binds to calcium-dependent protein kinase. In vitro phosphorylation assays showed that GmSerat2;1, but not GmSerat2;1 mutants (S378A or S378D), were phosphorylated by soybean calcium-dependent protein kinase isoforms.

Plasmodesmal-associated protein kinase in tobacco and Arabidopsis recognizes a subset of non-cell-autonomous proteins.

Cell-to-cell communication in plants involves the trafficking of macromolecules through specialized intercellular organelles, termed plasmodesmata. This exchange of proteins and RNA is likely regulated, and a role for protein phosphorylation has been implicated, but specific components remain to be identified. Here, we describe the molecular characterization of a plasmodesmal-associated protein kinase (PAPK).

A systemic small RNA signaling system in plants.

Systemic translocation of RNA exerts non-cell-autonomous control over plant development and defense. Long-distance delivery of mRNA has been proven, but transport of small interfering RNA and microRNA remains to be demonstrated. Analyses performed on phloem sap collected from a range of plants identified populations of small RNA species.

Selective trafficking of non-cell-autonomous proteins mediated by NtNCAPP1.

In plants, cell-to-cell communication is mediated by plasmodesmata and involves the trafficking of non-cell-autonomous proteins (NCAPs). A component in this pathway, Nicotiana tabacum NON-CELL-AUTONOMOUS PATHWAY PROTEIN1 (NtNCAPP1), was affinity purified and cloned. Protein overlay assays and in vivo studies showed that NtNCAPP1 is located on the endoplasmic reticulum at the cell periphery and displays specificity in its interaction with NCAPs.

Analysis of the complexity of protein kinases within the phloem sieve tube system. Characterization of Cucurbita maxima calmodulin-like domain protein kinase 1.

In angiosperms, functional, mature sieve elements lack nuclei, vacuoles, ribosomes, and most of the endomembrane network. In this study, the complexity, number, and nature of protein kinases within the phloem sap of Cucurbita maxima were investigated to test the hypothesis that the enucleate sieve tube system utilizes a simplified signal transduction network. Supporting evidence was obtained in that only five putative protein kinases (three calcium-independent and two calcium-dependent protein kinases) were detected within the phloem sap extracted from stem tissues.