Preclinical safety assessment of MV-s-NAP, a novel oncolytic measles virus strain armed with an H . pylori immunostimulatory bacterial transgene.

Despite recent therapeutic advances, metastatic breast cancer (MBC) remains incurable. Engineered measles virus (MV) constructs based on the attenuated MV Edmonston vaccine platform have demonstrated significant oncolytic activity against solid tumors. The Helicobacter pylori neutrophil-activating protein (NAP) is responsible for the robust inflammatory reaction in gastroduodenal mucosa during bacterial infection. NAP attracts and activates immune cells at the site of infection, inducing expression of pro-inflammatory mediators. We engineered an MV strain to express the secretory form of NAP (MV-s-NAP) and showed that it exhibits anti-tumor and immunostimulatory activity in human breast cancer xenograft models. In this study, we utilized a measles-infection-permissive mouse model (transgenic IFNAR KO-CD46Ge) to evaluate the biodistribution and safety of MV-s-NAP. The primary objective was to identify potential toxic side effects and confirm the safety of the proposed clinical doses of MV-s-NAP prior to a phase I clinical trial of intratumoral administration of MV-s-NAP in patients with MBC. Both subcutaneous delivery (corresponding to the clinical trial intratumoral administration route) and intravenous (worst case scenario) delivery of MV-s-NAP were well tolerated: no significant clinical, laboratory or histologic toxicity was observed. This outcome supports the safety of MV-s-NAP for oncolytic virotherapy of MBC. The first-in-human clinical trial of MV-s-NAP in patients with MBC (ClinicalTrials.gov: NCT04521764) was subsequently activated.

A brief review of reporter gene imaging in oncolytic virotherapy and gene therapy.

Reporter gene imaging (RGI) can accelerate development timelines for gene and viral therapies by facilitating rapid and noninvasive in vivo studies to determine the biodistribution, magnitude, and durability of viral gene expression and/or virus infection. Functional molecular imaging systems used for this purpose can be divided broadly into deep-tissue and optical modalities. Deep-tissue modalities, which can be used in animals of any size as well as in human subjects, encompass single photon emission computed tomography (SPECT), positron emission tomography (PET), and functional/molecular magnetic resonance imaging (f/mMRI). Optical modalities encompass fluorescence, bioluminescence, Cerenkov luminescence, and photoacoustic imaging and are suitable only for small animal imaging. Here we discuss the mechanisms of action and relative merits of currently available reporter gene systems, highlighting the strengths and weaknesses of deep tissue versus optical imaging systems and the hardware/reagents that are used for data capture and processing. In light of recent technological advances, falling costs of imaging instruments, better availability of novel radioactive and optical tracers, and a growing realization that RGI can give invaluable insights across the entire in vivo translational spectrum, the approach is becoming increasingly essential to facilitate the competitive development of new virus- and gene-based drugs.

Improved Noninvasive In Vivo Tracking of AAV-9 Gene Therapy Using the Perchlorate-Resistant Sodium Iodide Symporter from Minke Whale.

The sodium iodide symporter (NIS) is widely used as a reporter gene to noninvasively monitor the biodistribution and durability of vector-mediated gene expression via gamma scintigraphy, single-photon emission computed tomography (SPECT), and positron-emission tomography (PET). However, the approach is limited by background signal due to radiotracer uptake by endogenous NIS-expressing tissues. In this study, using the SPECT tracer pertechnetate (99mTcO4) and the PET tracer tetrafluoroborate (B18F4), in combination with the NIS inhibitor perchlorate, we compared the transport properties of human NIS and minke whale (Balaenoptera acutorostrata scammoni) NIS in vitro and in vivo. Based on its relative resistance to perchlorate, the NIS protein from minke whale appeared to be the superior candidate reporter gene. SPECT and PET imaging studies in nude mice challenged with NIS-encoding adeno-associated virus (AAV)-9 vectors confirmed that minke whale NIS, in contrast to human and endogenous mouse NIS, continues to function as a reliable reporter even when background radiotracer uptake by endogenous NIS is blocked by perchlorate.

Mapping of Ion and Substrate Binding Sites in Human Sodium Iodide Symporter (hNIS).

The human sodium iodide symporter (hNIS) is a theranostic reporter gene which concentrates several clinically approved SPECT and PET radiotracers and plays an essential role for the synthesis of thyroid hormones as an iodide transporter in the thyroid gland. Development of hNIS mutants which could enhance translocation of the desired imaging ions is currently underway. Unfortunately, it is hindered by lack of understanding of the 3D organization of hNIS and its relation to anion transport. There are no known crystal structures of hNIS in any of its conformational states. Homology modeling can be very effective in such situations; however, the low sequence identity between hNIS and relevant secondary transporters with available experimental structures makes the choice of a template and the generation of 3D models nontrivial. Here, we report a combined application of homology modeling and molecular dynamics refining of the hNIS structure in its semioccluded state. The modeling was based on templates from the LeuT-fold protein family and was done with emphasis on the refinement of the substrate-ion binding pocket. The consensus model developed in this work is compared to available biophysical and biochemical experimental data for a number of different LeuT-fold proteins. Some functionally important residues contributing to the formation of putative binding sites and permeation pathways for the cotransported Na+ ions and I- substrate were identified. The model predictions were experimentally tested by generation of mutant versions of hNIS and measurement of relative (to WT hNIS) 125I- uptake of 35 hNIS variants.

Inter-species variation in monovalent anion substrate selectivity and inhibitor sensitivity in the sodium iodide symporter (NIS).

The sodium iodide symporter (NIS) transports iodide, which is necessary for thyroid hormone production. NIS also transports other monovalent anions such as tetrafluoroborate (BF4-), pertechnetate (TcO4-), and thiocyanate (SCN-), and is competitively inhibited by perchlorate (ClO4-). However, the mechanisms of substrate selectivity and inhibitor sensitivity are poorly understood. Here, a comparative approach was taken to determine whether naturally evolved NIS proteins exhibit variability in their substrate transport properties. The NIS proteins of thirteen animal species were initially assessed, and three species from environments with differing iodide availability, freshwater species Danio rerio (zebrafish), saltwater species Balaenoptera acutorostrata scammoni (minke whale), and non-aquatic mammalian species Homo sapiens (human) were studied in detail. NIS genes from each of these species were lentivirally transduced into HeLa cells, which were then characterized using radioisotope uptake assays, 125I- competitive substrate uptake assays, and kinetic assays. Homology models of human, minke whale and zebrafish NIS were used to evaluate sequence-dependent impact on the organization of Na+ and I- binding pockets. Whereas each of the three proteins that were analyzed in detail concentrated iodide to a similar degree, their sensitivity to perchlorate inhibition varied significantly: minke whale NIS was the least impacted by perchlorate inhibition (IC50 = 4.599 µM), zebrafish NIS was highly sensitive (IC50 = 0.081 µM), and human NIS showed intermediate sensitivity (IC50 = 1.566 µM). Further studies with fifteen additional substrates and inhibitors revealed similar patterns of iodide uptake inhibition, though the degree of 125I- uptake inhibition varied with each compound. Kinetic analysis revealed whale NIS had the lowest Km-I and the highest Vmax-I. Conversely, zebrafish NIS had the highest Km and lowest Vmax. Again, human NIS was intermediate. Molecular modeling revealed a high degree of conservation in the putative ion binding pockets of NIS proteins from different species, which suggests the residues responsible for the observed differences in substrate selectivity lie elsewhere in the protein. Ongoing studies are focusing on residues in the extracellular loops of NIS as determinants of anion specificity. These data demonstrate significant transport differences between the NIS proteins of different species, which may be influenced by the unique physiological needs of each organism. Our results also identify naturally-existing NIS proteins with significant variability in substrate transport kinetics and inhibitor sensitivity, which suggest that the affinity and selectivity of NIS for certain substrates can be altered for biotechnological and clinical applications. Further examination of interspecies differences may improve understanding of the substrate transport mechanism.

Heat induction of a novel Rad9 variant from a cryptic translation initiation site reduces mitotic commitment.

Exposure of human cells to heat switches the activating signal of the DNA damage checkpoint from genotoxic to temperature stress. This change reduces mitotic commitment at the expense of DNA break repair. The thermal alterations behind this switch remain elusive despite the successful use of heat to sensitise cancer cells to DNA breaks. Rad9 is a highly conserved subunit of the Rad9-Rad1-Hus1 (9-1-1) checkpoint-clamp that is loaded by Rad17 onto damaged chromatin. At the DNA, Rad9 activates the checkpoint kinases Rad3(ATR) and Chk1 to arrest cells in G2. Using Schizosaccharomyces pombe as a model eukaryote, we discovered a new variant of Rad9, Rad9-M50, whose expression is specifically induced by heat. High temperatures promote alternative translation from a cryptic initiation codon at methionine-50. This process is restricted to cycling cells and is independent of the temperature-sensing mitogen-activated protein kinase (MAPK) pathway. While full-length Rad9 delays mitosis in the presence of DNA lesions, Rad9-M50 functions in a remodelled checkpoint pathway to reduce mitotic commitment at elevated temperatures. This remodelled pathway still relies on Rad1 and Hus1, but acts independently of Rad17. Heat-induction of Rad9-M50 ensures that the kinase Chk1 remains in a hypo-phosphorylated state. Elevated temperatures specifically reverse the DNA-damage-induced modification of Chk1 in a manner dependent on Rad9-M50. Taken together, heat reprogrammes the DNA damage checkpoint at the level of Chk1 by inducing a Rad9 variant that can act outside of the canonical 9-1-1 complex.

Identification of neuronal substrates implicates Pak5 in synaptic vesicle trafficking.

Synaptic transmission is mediated by a complex set of molecular events that must be coordinated in time and space. While many proteins that function at the synapse have been identified, the signaling pathways regulating these molecules are poorly understood. Pak5 (p21-activated kinase 5) is a brain-specific isoform of the group II Pak kinases whose substrates and roles within the central nervous system are largely unknown. To gain insight into the physiological roles of Pak5, we engineered a Pak5 mutant to selectively radiolabel its substrates in murine brain extract. Using this approach, we identified two novel Pak5 substrates, Pacsin1 and Synaptojanin1, proteins that directly interact with one another to regulate synaptic vesicle endocytosis and recycling. Pacsin1 and Synaptojanin1 were phosphorylated by Pak5 and the other group II Paks in vitro, and Pak5 phosphorylation promoted Pacsin1-Synaptojanin1 binding both in vitro and in vivo. These results implicate Pak5 in Pacsin1- and Synaptojanin1-mediated synaptic vesicle trafficking and may partially account for the cognitive and behavioral deficits observed in group II Pak-deficient mice.

Evolution of tumor invasiveness: the adaptive tumor microenvironment landscape model.

Interactions between cancer cells and their microenvironment are crucial for promoting tumor growth and invasiveness. In the tumor adaptive landscape model, hypoxic and acidic microenvironmental conditions reduce the fitness of cancer cells and significantly restrict their proliferation. This selects for enhanced motility as cancer cells may evolve an invasive phenotype if the consequent cell movement is rewarded by proliferation. Here, we used an integrative approach combining a mathematical tumor adaptive landscape model with experimental studies to examine the evolutionary dynamics that promote an invasive cancer phenotype. Computer simulation results hypothesized an explicit coupling of motility and proliferation in cancer cells. The mathematical modeling results were also experimentally examined by selecting Panc-1 cells with enhanced motility on a fibroblast-derived 3-dimensional matrix for cells that move away from the unfavorable metabolic constraints. After multiple rounds of selection, the cells that adapted through increased motility were characterized for their phenotypic properties compared with stationary cells. Microarray and gene depletion studies showed the role of Rho-GDI2 in regulating both cell movement and proliferation. Together, this work illustrates the partnership between evolutionary mathematical modeling and experimental validation as a potentially useful approach to study the complex dynamics of the tumor microenvironment.