Phase I clinical trial of systemically administered TUSC2(FUS1)-nanoparticles mediating functional gene transfer in humans

Background

Tumor suppressor gene TUSC2/FUS1 (TUSC2) is frequently inactivated early in lung cancer development. TUSC2 mediates apoptosis in cancer cells but not normal cells by upregulation of the intrinsic apoptotic pathway. No drug strategies currently exist targeting loss-of–function genetic abnormalities. We report the first in-human systemic gene therapy clinical trial of tumor suppressor gene TUSC2.

Methods

Patients with recurrent and/or metastatic lung cancer previously treated with platinum-based chemotherapy were treated with escalating doses of intravenous N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP):cholesterol nanoparticles encapsulating a TUSC2 expression plasmid (DOTAP:chol-TUSC2) every 3 weeks.

Results

Thirty-one patients were treated at 6 dose levels (range 0.01 to 0.09 milligrams per kilogram). The MTD was determined to be 0.06 mg/kg. Five patients achieved stable disease (2.6–10.8 months, including 2 minor responses). One patient had a metabolic response on positron emission tomography (PET) imaging. RT-PCR analysis detected TUSC2 plasmid expression in 7 of 8 post-treatment tumor specimens but not in pretreatment specimens and peripheral blood lymphocyte controls. Proximity ligation assay, performed on paired biopsies from 3 patients, demonstrated low background TUSC2 protein staining in pretreatment tissues compared with intense (10–25 fold increase) TUSC2 protein staining in post-treatment tissues. RT-PCR gene expression profiling analysis of apoptotic pathway genes in two patients with high post-treatment levels of TUSC2 mRNA and protein showed significant post-treatment changes in the intrinsic apoptotic pathway. Twenty-nine genes of the 82 tested in the apoptosis array were identified by Igenuity Pathway Analysis to be significantly altered post-treatment in both patients (Pearson correlation coefficient 0.519; p<0.01).

Conclusions

DOTAP:chol-TUSC2 can be safely administered intravenously in lung cancer patients and results in uptake of the gene by human primary and metastatic tumors, transgene and gene product expression, specific alterations in TUSC2-regulated pathways, and anti-tumor effects (to our knowledge for the first time for systemic DOTAP:cholesterol nanoparticle gene therapy).

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00059605","term_id":"NCT00059605"}}NCT00059605     

Signaling from the secretory granule to the nucleus

Neurons and endocrine cells use a complex array of signaling molecules to communicate with each other and with various targets. The majority of these signaling molecules are stored in specialized organelles awaiting release on demand: 40-60 nm vesicles carry conventional or small molecule neurotransmitters, and 200-400 nm granules contain bioactive peptides. The supply of small molecule neurotransmitters is tightly regulated by local feedback of synthetic rates and transport processes at sites of release. The larger granules that contain bioactive peptides present the secretory cell with special challenges, as the peptide precursors are inserted into the lumen of the secretory pathway in the cell soma and undergo biosynthetic processing while being transported to distant sites for eventual secretion. One solution to this dilemma in information handling has been to employ proteolytic cleavage of secretory granule membrane proteins to produce cytosolic fragments that can signal to the nucleus, affecting gene expression. The use of regulated intramembrane proteolysis to signal from secretory granules to the nucleus is compared to its much better understood role in relaying information from the endoplasmic reticulum by SREBP and ATF6 and from the plasma membrane by cadherins, Notch and ErbB4.     

The covalent modification and regulation of TLR8 in HEK-293 cells stimulated with imidazoquinoline agonists

The mammalian TLRs (Toll-like receptors) mediate the rapid initial immune response to pathogens through recognition of pathogen-associated molecular patterns. The pathogen pattern to which TLR8 responds is ssRNA (single-stranded RNA) commonly associated with ssRNA viruses. TLR8 also responds to small, purine-like molecules including the imidazoquinoline IRMs (immune-response modifiers). The IRMs include molecules that selectively activate TLR7, selectively activate TLR8 or non-selectively activate both TLR7 and TLR8. Using HEK-293 cells (human embryonic kidney cells) stably expressing an NF-kappaB (nuclear factor kappaB)/luciferase promoter-reporter system as a model system, we have examined the regulation of TLR8 using the non-selective TLR7/8 agonist, 3M-003. Using conservative tyrosine to phenylalanine site-directed mutation, we show that of the 13 tyrosine residues resident in the cytosolic domain of TLR8, only three appear to be critical to TLR8 signalling. Two of these, Tyr898 and Tyr904, reside in the Box 1 motif and the third, Tyr1048, lies in a YXXM putative p85-binding motif. TLR8 is tyrosine-phosphorylated following 3M-003 treatment and TLR8 signalling is inhibited by tyrosine kinase inhibitors. Treatment with 3M-003 results in the association of the p85 regulatory subunit of PI3K (phosphoinositide 3-kinase) with TLR8 and this association is inhibited by tyrosine to phenylalanine mutation of either the YXXM or Box 1 motifs. As a further consequence of activation by 3M-003, TLR8 is modified to yield both higher and lower molecular mass species. These species include a monoubiquitinated form as deduced from ubiquitin peptide sequencing by HPLC/MS/MS (tandem MS).     

Changes in the level of cytosolic calcium, nitric oxide and nitric oxide synthase activity during platelet aggregation: an in vitro study in platelets from normal subjects and those with cirrhosis

Variceal bleeding due to abnormal platelet function is a well-known complication of cirrhosis. Nitric oxide-related stress has been implicated in the pathogenesis of liver cirrhosis.In the present investigation,we evaluated the level of platelet aggregation and concomitant changes in the level of platelet cytosolic calcium (Ca2+), nitric oxide (NO) and NO synthase (NOS) activity in liver cirrhosis.The aim of the present study was to investigate whether the production of NO by NOS and level of cytosolic Ca2+ influence the aggregation of platelets in patients with cirrhosis of the liver.Agonist-induced aggregation and the simultaneous changes in the level of cytosolic Ca2+, NO and NOS were monitored in platelets of patients with cirrhosis.Platelet aggregation was also measured in the presence of the eNOS inhibitor,diphenylene iodinium chloride (DIC).The level of agonist-induced platelet aggregation was significantly low in the platelets of patients with cirrhosis compared with that in platelets from normal subjects.During the course of platelet aggregation,concomitant elevation in the level of cytosolic Ca2+ was observed in normal samples,whereas the elevation was not significant in platelets of patients with cirrhosis.A parallel increase was observed in the levels of NO and NOS activity.In the presence of the eNOS inhibitor,platelet aggregation was enhanced and accompanied by an elevated calcium level.The inhibition of platelet aggregation in liver cirrhosis might be partly due to greater NO formation by eNOS.Defective Ca2+ release from the internal stores to the cytosol may account for inhibition of aggregation of platelets in cirrhosis.The NO-related defective aggregation of platelets in patients with cirrhosis found in our study is of clinical importance,and the underlying mechanism of such changes suggests a possible therapeutic strategy with cell-specific NO blockers.     

Comparative chlorine and temperature tolerance of the oyster crassostrea madrasensis: Implications for cooling system fouling

Crassostrea madrasensis is an important fouling oyster in tropical industrial cooling water systems. C. madrasensis individuals attach to surfaces by cementing one of their two valves to the substratum. Therefore, oyster fouling creates more problems than mussel fouling in the cooling conduits of power stations, because unlike the latter, the shell of the former remains attached to the substratum even after the death of the animal. However, there are no published reports on the tolerance of this species to chlorination and heat treatment. The mortality pattern and physiological behaviour (oxygen consumption and filtration rate) of three size groups (13 mm, 44 mm and 64 mm mean shell length) of C. madrasensis were studied at different residual chlorine concentrations (0.25, 0.5, 0.75, 1, 2, 3 to 5 mg l^{m 1}) and temperatures (30°C to 45°C). The effect of shell size (=age) on C. madrasensis mortality in the presence of chlorine and taking into account temperature was significant, with the largest size group oysters showing highest resistance. At 1 mg l^{m 1} residual chlorine, the 13 mm and 64 mm size group oysters took 504 h (21 d) and 744 h (31 d), respectively to reach 100% mortality. At 39°C, the 13 mm size group oysters took 218 min to reach 100% mortality, whereas the 64 mm size group oysters took 325 min. The oxygen consumption and filtration rate of C. madrasensis showed progressive reduction with increasing residual chlorine concentrations. However, the filtration rate and oxygen consumption responses of C. madrasensis were not significantly different between 30°C (control) and 37.5°C. There was a sharp decrease in the filtration rate and oxygen consumption at 38.5°C. A comparison of the present mortality data with previous reports on other bivalves suggests that the chlorine tolerance of C. madrasensis lies in between that of Perna viridis and Perna perna, while its temperature tolerance is significantly higher than that of the other two bivalve species. However, in power station heat exchangers, where simultaneous chlorine and thermal stresses are existent, C. madrasensis may have an edge over other common foulants, because of its high temperature tolerance.     

Papain Does Not Cleave Operator-Bound Lambda Repressor: Structural Characterization of the Carboxy Terminal Domain and the Hinge

Abstract

The circular dichroism spectra of three different purified carboxy terminal fragments 93–236, 112–236 and 132–236 of the bacteriophage γ cI repressor have been measured and compared with those of the intact repressor and the amino terminal fragment 1–92. All three carboxy terminal fragments contain mostly β-strands and loops, a minor helix content increasing with the size of the fragment, showing that the 93–131 region previously called a hinge is structured. Fourier transformed infrared spectra also showed that fragment 93–236 contains α-helices, β-sheets and turns but fragment 132–236 contains no detectable α-helix, only β-sheets and turns. Papain is known to cleave the γ repressor, but it is shown here that it cannot cleave the operator-bound repressor dimer. For the 132–236 fragment, both the wt and the SN228 mutant previously shown to be dimerization defective in the intact, gave similar dimerization properties as investigated by HPLC at 2 to 100 µM protein concentration, with a KD of 13.2 µM and 19.1 µM respectively. The papain cleavage for wt and SN228 proceed at equal rates for the first cleavage at 92–93; however, the subsequent cleavages are faster for SN228. The three Cys residues in the 132–236 fragment were found to be unreactive upon incubation with DTNB, indicating the thiol sulfur atoms are buried in the repressor carboxy terminal domain. Denaturation of the 132–236 fragment studied by tryptophan fluorescence shows two transitions centered at 1.5 M and 4.5 M of urea.     

Intracellular mediators of transforming growth factor β superfamily signaling localize to endosomes in chicken embryo and mouse lenses in vivo

Background  

Endocytosis is a key regulator of growth factor signaling pathways. Recent studies showed that the localization to endosomes of intracellular mediators of growth factor signaling may be required for their function. Although there is substantial evidence linking endocytosis and growth factor signaling in cultured cells, there has been little study of the endosomal localization of signaling components in intact tissues or organs.