A Phase II Study of Pelareorep (REOLYSIN®) in Combination with Gemcitabine for Patients with Advanced Pancreatic Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis, with 1 and 5-year survival rates of ~18% and 7% respectively. FOLFIRINOX or gemcitabine in combination with nab-paclitaxel are standard treatment options for metastatic disease. However, both regimens are more toxic than gemcitabine alone. Pelareorep (REOLYSIN®), a proprietary isolate of reovirus Type 3 Dearing, has shown antitumor activity in clinical and preclinical models. In addition to direct cytotoxic effects, pelareorep can trigger antitumor immune responses. Due to the high frequency of RAS mutations in PDAC, we hypothesized that pelareorep would promote selective reovirus replication in pancreatic tumors and enhance the anticancer activity of gemcitabine. Chemotherapy-naïve patients with advanced PDAC were eligible for the study. The primary objective was Clinical Benefit Rate (complete response (CR) + partial response (PR) + stable disease (SD) ≥ 12 weeks) and secondary objectives include overall survival (OS), toxicity, and pharmacodynamics (PD) analysis. The study enrolled 34 patients; results included one partial response, 23 stable disease, and 5 progressive disease. The median OS was 10.2 months, with a 1- and 2-year survival rate of 45% and 24%, respectively. The treatment was well tolerated with manageable nonhematological toxicities. PD analysis revealed reovirus replication within pancreatic tumor and associated apoptosis. Upregulation of immune checkpoint marker PD-L1 suggests future consideration of combining oncolytic virus therapy with anti-PD-L1 inhibitors. We conclude that pelareorep complements single agent gemcitabine in PDAC.

A phase II study of REOLYSIN® (pelareorep) in combination with carboplatin and paclitaxel for patients with advanced malignant melanoma.

REOLYSIN® (pelareorep) is an investigational new drug, consisting of a live, replication-competent, Reovirus Type 3 Dearing strain in a proprietary formulation. Several preclinical and clinical trials with REOLYSIN® on a wide range of cancer indications have demonstrated antineoplastic activity on cells with activated RAS-signaling pathway. Furthermore, long-term survival benefits were evident in post-treatment patients indicating a potential antitumor immune response triggered by REOLYSIN®. Numerous mono and/or combination therapy studies with the agent showed a consistent safety profile. The current study is a phase II, single-arm, open label trial of REOLYSIN® in combination with carboplatin and paclitaxel for patients with advanced melanoma. Results from the 14 patients enrolled in the study exhibited no grade 4 adverse events or deaths but manageable grade-3 toxicities commonly attributed to REOLYSIN®, including pyrexia, chills, myalgia, pain, fatigue, and nausea. The number of treatment cycles ranged from 2 to 20 with a median of 6 cycles. The study met its treatment and efficacy goal for the first stage with three partial responses (ORR was 21%). No complete responses were noted. The median PFS and OS were 5.2 and 10.9 months, respectively. The 1-year OS was 43% with a disease control rate of 85%. In conclusion, REOLYSIN® combined with carboplatin and paclitaxel is a safe and potentially efficacious therapy for patients with advanced malignant melanoma. Additional combination studies using REOLYSIN® with chemo/immunotherapy drugs may support more favorable outcomes for patients in this indication.

The oncolytic virus, pelareorep, as a novel anticancer agent: a review.

Pelareorep (REOLYSIN®) is an investigational new drug, a proprietary formulation consisting of a live, replication-competent, naturally occurring Reovirus Type 3 Dearing strain. Through several preclinical studies it was determined that reovirus can exhibit profound cytotoxic effects on cancer cells predominantly with an activated RAS-signalling pathway. Moreover, it was discovered that reoviruses can "hitchhike" on peripheral blood mononuclear cells and dendritic cells, thereby evading neutralizing antibodies of the host immune system. Cell carriage, targeted delivery, triggering host immune response and other inherent characteristics of the reovirus led to its further advancement into cancer therapy. When injected into Sprague-Dawley rats, the viral routes of clearance, predominantly through the spleen and liver, remained consistent with earlier studies. Toxicology findings were considered incidental and not associated with pelareorep when tested in animal models. Pelareorep demonstrated a high level of homogeneity at the amino acid level and genetic stability when compared to the master and working virus banks. The drug is manufactured in a 100 L bioreactor after which it is purified and formulated for use in pre-clinical, clinical and research studies. Over the past few decades, we have witnessed a paradigm shift from conventional therapy to the conceivable use of oncolytic viruses for the treatment of cancer. This review will detail pre-clinical evidence of anticancer activity of pelareorep that has led to extensive clinical development. Several Phase I-II clinical trials have been completed or are ongoing in cancer patients on a broad spectrum of solid tumors and hematologic malignancies.

Silencing the lettuce homologs of small rubber particle protein does not influence natural rubber biosynthesis in lettuce (Lactuca sativa).

Natural rubber, cis-1,4-polyisoprene, is an important raw material in chemical industries, but its biosynthetic mechanism remains elusive. Natural rubber is known to be synthesized in rubber particles suspended in laticifer cells in the Brazilian rubber tree (Hevea brasiliensis). In the rubber tree, rubber elongation factor (REF) and its homolog, small rubber particle protein (SRPP), were found to be the most abundant proteins in rubber particles, and they have been implicated in natural rubber biosynthesis. As lettuce (Lactuca sativa) can synthesize natural rubber, we utilized this annual, transformable plant to examine in planta roles of the lettuce REF/SRPP homologs by RNA interference. Among eight lettuce REF/SRPP homologs identified, transcripts of two genes (LsSRPP4 and LsSRPP8) accounted for more than 90% of total transcripts of REF/SRPP homologs in lettuce latex. LsSRPP4 displays a typical primary protein sequence as other REF/SRPP, while LsSRPP8 is twice as long as LsSRPP4. These two major LsSRPP transcripts were individually and simultaneously silenced by RNA interference, and relative abundance, polymer molecular weight, and polydispersity of natural rubber were analyzed from the LsSRPP4- and LsSRPP8-silenced transgenic lettuce. Despite previous data suggesting the implications of REF/SRPP in natural rubber biosynthesis, qualitative and quantitative alterations of natural rubber could not be observed in transgenic lettuce lines. It is concluded that lettuce REF/SRPP homologs are not critically important proteins in natural rubber biosynthesis in lettuce.

A lettuce (Lactuca sativa) homolog of human Nogo-B receptor interacts with cis-prenyltransferase and is necessary for natural rubber biosynthesis.

Natural rubber (cis-1,4-polyisoprene) is an indispensable biopolymer used to manufacture diverse consumer products. Although a major source of natural rubber is the rubber tree (Hevea brasiliensis), lettuce (Lactuca sativa) is also known to synthesize natural rubber. Here, we report that an unusual cis-prenyltransferase-like 2 (CPTL2) that lacks the conserved motifs of conventional cis-prenyltransferase is required for natural rubber biosynthesis in lettuce. CPTL2, identified from the lettuce rubber particle proteome, displays homology to a human NogoB receptor and is predominantly expressed in latex. Multiple transgenic lettuces expressing CPTL2-RNAi constructs showed that a decrease of CPTL2 transcripts (3-15% CPTL2 expression relative to controls) coincided with the reduction of natural rubber as low as 5%. We also identified a conventional cis-prenyltransferase 3 (CPT3), exclusively expressed in latex. In subcellular localization studies using fluorescent proteins, cytosolic CPT3 was relocalized to endoplasmic reticulum by co-occurrence of CPTL2 in tobacco and yeast at the log phase. Furthermore, yeast two-hybrid data showed that CPTL2 and CPT3 interact. Yeast microsomes containing CPTL2/CPT3 showed enhanced synthesis of short cis-polyisoprenes, but natural rubber could not be synthesized in vitro. Intriguingly, a homologous pair CPTL1/CPT1, which displays ubiquitous expressions in lettuce, showed a potent dolichol biosynthetic activity in vitro. Taken together, our data suggest that CPTL2 is a scaffolding protein that tethers CPT3 on endoplasmic reticulum and is necessary for natural rubber biosynthesis in planta, but yeast-expressed CPTL2 and CPT3 alone could not synthesize high molecular weight natural rubber in vitro.

Evaluation of homogeneity and genetic stability of REOLYSIN (pelareorep) by complete genome sequencing of reovirus after large scale production.

REOLYSIN (pelareorep) is a proprietary isolate of the reovirus T3D (Type 3 Dearing) strain which is currently being tested in clinical trials as an anticancer therapeutic agent. Reovirus genomes are composed of ten segments of double-stranded ribonucleic acid (RNA) characterized by genome size: large (L1, L2, and L3), medium (M1, M2, and M3), and small (S1, S2, S3, and S4). The objective of this work was to evaluate the homogeneity and genetic stability of REOLYSIN. Sanger sequencing (SS) performed on test articles derived from the Master Virus Bank (MVB) and Working Virus Bank (WVB) identified many modifications when compared to GenBank reference sequences. Massively parallel sequencing (MPS) using Roche-454 sequencing was performed on REOLYSIN (100 L scale) and resulted in 69,821,115 bases and an average of 335 bases per read. Twenty-nine high confidence differences relative to the GenBank reference sequence were identified in REOLYSIN by MPS. Of those, 27 were previously identified by SS in the virus bank-derived test articles. Of the remaining two nucleotide differences, one was predicted to be silent at the amino acid level (L3 genome-T3163C, codon 1054, 86% of the population was "T" and 13% of the population were reported as "C"). The other modification was in the noncoding region (M1 genome-A2284A to A2284G), and A2284G was present in 97% of the population. The results obtained from MPS were comparable to those from SS; both demonstrate a high level of homogeneity at the amino acid level and genetic stability of REOLYSIN. Finally, phylogenetic analysis of the REOLYSIN L1 genome segment showed close evolutionary relationship with its human homologs, serotypes Lang and Dearing.

Bio-distribution study of Reolysin® (pelareorep) through a single intravenous infusion in Sprague-Dawley rats.

Numerous pre-clinical and clinical studies on reovirus have generated valuable information which supports the use of this orphan virus as an investigational drug for cancer treatment. Reolysin® (pelareorep) is a clinical formulation of the human Reovirus Type 3 Dearing strain. The clinical safety and efficacy of Reolysin® in humans is being tested on an assortment of cancer indications as a mono and/or combination therapy. Reovirus has many inherent characteristics that make it a potential candidate for virotherapy, including: the rapid and natural spread through the haematogenous route, the ability to overcome immunological barriers thereby reaching tumor sites, and being replication-competent. The purpose of this study was to elucidate the bio-distribution pattern of Reolysin® in healthy Sprague-Dawley rats. Following a single 15-min intravenous infusion via the tail vein in Sprague-Dawley rats, the levels of virus genome were determined in 16 organs/tissues by RT-qPCR (Reverse Transcriptase- Quantitative Polymerase Chain Reaction) over a 336 h (Day 15) incubation regime. Consistent with previous studies, maximal reovirus RNA levels were observed in the spleen; indicating its involvement in viral uptake and clearance, followed by heart, ovaries, tail (infusion site), liver and lungs. All the organs/tissues demonstrated unquantifiable levels of reovirus genome at the end of incubation, suggesting substantial to complete viral clearance. Several studies in the last decade have described the use of reovirus for treating ovarian cancers. An increase of reovirus genome in ovaries at 24 h post infection was noted. The results will aid in the design of additional exploratory clinical trials for Reolysin®.

Transcriptome analysis based on next-generation sequencing of non-model plants producing specialized metabolites of biotechnological interest.

Plants produce a vast array of specialized metabolites, many of which are used as pharmaceuticals, flavors, fragrances, and other high-value fine chemicals. However, most of these compounds occur in non-model plants for which genomic sequence information is not yet available. The production of a large amount of nucleotide sequence data using next-generation technologies is now relatively fast and cost-effective, especially when using the latest Roche-454 and Illumina sequencers with enhanced base-calling accuracy. To investigate specialized metabolite biosynthesis in non-model plants we have established a data-mining framework, employing next-generation sequencing and computational algorithms, to construct and analyze the transcriptomes of 75 non-model plants that produce compounds of interest for biotechnological applications. After sequence assembly an extensive annotation approach was applied to assign functional information to over 800,000 putative transcripts. The annotation is based on direct searches against public databases, including RefSeq and InterPro. Gene Ontology (GO), Enzyme Commission (EC) annotations and associated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway maps are also collected. As a proof-of-concept, the selection of biosynthetic gene candidates associated with six specialized metabolic pathways is described. A web-based BLAST server has been established to allow public access to assembled transcriptome databases for all 75 plant species of the PhytoMetaSyn Project (www.phytometasyn.ca).

Myrosinases from root and leaves of Arabidopsis thaliana have different catalytic properties.

Myrosinases (EC 3.2.1.147) are beta-thioglucoside glucosidases present in Brassicaceae plants. These enzymes serve to protect plants against pathogens and insect pests by initiating breakdown of the secondary metabolites glucosinolates into toxic products. Several forms of myrosinases are present in plants but the properties and role of different isoenzymes are not well understood. The dicot plant model organism Arabidopsis thaliana seems to contain six myrosinase genes (TGG1-TGG6). In order to compare the different myrosinases, cDNAs corresponding to TGG1 from leaves and TGG4 and TGG5 from roots were cloned and overexpressed in Pichia pastoris. The His-tagged recombinant proteins were purified using affinity chromatography and the preparations were homogenous according to SDS-PAGE analysis. Myrosinase activity was confirmed for all forms and compared with respect to catalytic activity towards the allyl-glucosinolate sinigrin. There was a 22-fold difference in basal activity among the myrosinases. The enzymes were active in a broad pH range, are rather thermostable and active in a wide range of salt concentrations but sensitive to high salt concentrations. The myrosinases showed different activation-inhibition responses towards ascorbic acid with maximal activity around 0.7-1 mM. No activity was registered towards desulphosinigrin and this compound did not inhibit myrosinase activity towards sinigrin. All myrosinases also displayed O-beta-glucosidase activity, although with lower efficiency compared to the myrosinase activity. The differences in catalytic properties among myrosinase isozymes for function in planta are discussed.

Transient expression in Nicotiana benthamiana fluorescent marker lines provides enhanced definition of protein localization, movement and interactions in planta.

Here, we report on the construction of a novel series of Gateway-compatible plant transformation vectors containing genes encoding autofluorescent proteins, including Cerulean, Dendra2, DRONPA, TagRFP and Venus, for the expression of protein fusions in plant cells. To assist users in the selection of vectors, we have determined the relative in planta photostability and brightness of nine autofluorescent proteins (AFPs), and have compared the use of DRONPA and Dendra2 in photoactivation and photoconversion experiments. Additionally, we have generated transgenic Nicotiana benthamiana lines that express fluorescent protein markers targeted to nuclei, endoplasmic reticulum or actin filaments. We show that conducting bimolecular fluorescence complementation assays in plants that constitutively express cyan fluorescent protein fused to histone 2B provides enhanced data quality and content over assays conducted without the benefit of a subcellular marker. In addition to testing protein interactions, we demonstrate that our transgenic lines that express red fluorescent protein markers offer exceptional support in experiments aimed at defining nuclear or endomembrane localization. Taken together, the new combination of pSITE-BiFC and pSITEII vectors for studying intracellular protein interaction, localization and movement, in conjunction with our transgenic marker lines, constitute powerful tools for the plant biology community.

Membrane and protein dynamics in virus-infected plant cells.

In terms of functional genomics research, Nicotiana benthamiana, more so than other model plants, is highly amenable to high-throughput methods, especially those employing virus-induced gene silencing and agroinfiltration. Furthermore, through recent and ongoing sequencing projects, there are now upward of 18,000 unique N. benthamiana ESTs to support functional genomics research. Despite these advances, the cell biology of N. benthamiana itself, and in the context of virus infection, lags behind that of other model systems. Therefore, to meet the challenges of diverse cell biology studies that will be derived from ongoing functional genomics projects, a series of methods relevant to the characterization of membrane and protein dynamics in virus-infected cells are provided here. The data presented here were derived from our studies with plant rhabdoviruses. However, the employed techniques should be broadly applicable within the field of plant virology. We report here on the use of a novel series of binary vectors for the transient or stable expression of autofluorescent protein fusions in plants. Use of these vectors in conjunction with advanced microscopy techniques such as fluorescent recovery after photobleaching and total internal fluorescence microscopy, has revealed novel insight into the membrane and protein dynamics of virus-infected cells.

PSITE vectors for stable integration or transient expression of autofluorescent protein fusions in plants: probing Nicotiana benthamiana-virus interactions.

Plant functional proteomics research is increasingly dependent upon vectors that facilitate high-throughput gene cloning and expression of fusions to autofluorescent proteins. Here, we describe the pSITE family of plasmids, a new set of Agrobacterium binary vectors, suitable for the stable integration or transient expression of various autofluorescent protein fusions in plant cells. The pSITE vectors permit single-step Gateway-mediated recombination cloning for construction of binary vectors that can be used directly in transient expression studies or for the selection of transgenic plants on media containing kanamycin. These vectors can be used to express native proteins or fusions to monmeric red fluorescent protein or the enhanced green fluorescent protein and its cyan and yellow-shifted spectral variants. We have validated the vectors for use in transient expression assays and for the generation of transgenic plants. Additionally, we have generated markers for fluorescent highlighting of actin filaments, chromatin, endoplasmic reticulum, and nucleoli. Finally, we show that pSITE vectors can be used for targeted gene expression in virus-infected cells, which should facilitate high-throughput characterization of protein dynamics in host-virus interactions.

Membrane and protein dynamics in live plant nuclei infected with Sonchus yellow net virus, a plant-adapted rhabdovirus.

Sonchus yellow net virus (SYNV) serves as the paradigm for the cell biology of plant-adapted rhabdoviruses. Fluorescence recovery after photobleaching (FRAP) demonstrated that SYNV-induced intranuclear membranes are contiguous with the endomembrane system. Fluorescence intensity measurements of a green fluorescent protein-tagged nuclear envelope marker were consistent with electron microscopy studies, which suggest that infection by SYNV results in invagination of the inner nuclear membrane. Fusions of a red fluorescent protein to five SYNV-encoded proteins were used to determine the relationship between virus-induced intranuclear membranes and the localization of viral proteins. These data establish definitively that localization in the context of infected cells provides a superior means to predict protein function compared with localization studies conducted in mock-inoculated cells. Substructure has been identified within the viroplasm, the putative site of virus replication, which suggests that the nucleocapsid (N) protein occupies a region at the junction between the viroplasm and intranuclear membranes that largely excludes the phosphoprotein. Within virus-infected nuclei, the SYNV matrix (M) protein and glycoprotein (G) were associated predominantly with membranes, whereas sc4, the predicted movement protein, accumulated primarily at punctate loci on the periphery of cells. Coexpression of differently tagged SYNV protein fusions in combination with FRAP analyses suggest a model whereby the replication and morphogenesis of SYNV are spatially separated events. Finally, an M protein-containing complex was discovered that appears to bud from the nucleus and that moves on ER membranes. Taken together, these data represent the most comprehensive analyses of rhabdoviral protein localization conducted in the context of infected cells.

Occurrence of trypsin-like protease in cardamom (Elettaria cardamomum Maton).

Occurrence of trypsin-like protease in fresh cardamom (Elettaria cardamomum Maton) seeds, as evidenced by the benzoyl-arg-p-nitroanilide (BApNA) hydrolyzing ability of the seed enzyme preparation under alkaline condition is reported for the first time. The enzyme has a pH and temperature optima as 8 and 45 degrees C, respectively. It is inhibited by aprotinin and phenylmethyl sulfonyl fluoride (PMSF) in a dose-dependent manner, suggesting the presence of serine residues at the active site. The enzyme had a V(max) of 98.01 nmoles p-nitroaniline released per min per mg protein and K(m) of 0.0684 mM with BApNA as substrate. Addition of aprotinin (75.75 microM) increased K(m) value by three-folds, whereas the V(max) was reduced by 23%.