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     

Initiation of caspase-independent death in mouse mesangial cells by Cd2+: involvement of p38 kinase and CaMK-II

Cadmium (Cd) is a toxic metal with multiple effects on cell signaling and cell death. We studied the effects of Cd(2+) on quiescent mouse mesangial cells in serum-free conditions. Cadmium induces cell death over 6 h through annexin V+ states without or with causing uptake of propidium iodide, termed apoptotic and apoptosis-like death, respectively. Little or no necrosis is observed, and cell death is caspase-independent and associated with nuclear translocation of the apoptosis-inducing factor, AIF. We previously showed that Cd(2+) increased phosphorylation of Erk and CaMK-II, and CaMK-II activation increased cell death in an Erk-independent manner. Here we demonstrate that Cd(2+) increases Jnk and p38 kinase phosphorylation, and inhibition of p38-but not of Jnk-increases cell viability by suppressing apoptosis in preference to apoptosis-like death. Neither p38 kinase nor CaMK-II inhibition protects against a decrease in mitochondrial membrane potential, psi, indicating that kinase-mediated death is either independent of, or involves events downstream of a mitochondrial pathway. However, both the antioxidant N-acetyl cysteine (NAC) and the mitochondrial membrane-stabilizing agent cyclosporine A (CsA) partially preserve psi, suppress activation of p38 kinase, and partially protect the cells from Cd(2+)-induced death. Whereas the effect of CsA is on apoptosis, NAC acts on apoptosis-like death. Inhibition of glutathione synthesis exacerbates a Cd(2+)-dependent increase in cellular peroxides and favors apoptosis-like death over apoptosis. The caspase-independence of these modes of cell death is not due to an absence of this machinery in the mesangial cells: when they are exposed to Cd(2+) for longer periods in the presence of serum, procaspase-3 and PARP are cleaved and caspase inhibition is protective. We conclude that Cd(2+) can kill mesangial cells by multiple pathways, including caspase-dependent and -independent apoptotic and apoptosis-like death. Necrosis is not prominent. Activation of p38 kinase and of CaMK-II by Cd(2+) are associated with caspase-independent apoptosis that is not dependent on mitochondrial destabilization.     

Osmiophilic bodies and the odd organelles of alveolates

The apicomplexa are parasitic protozoa that are responsible for important human and animal diseases, including malaria, toxoplasmosis, cryptosporidiosis, coccidiosis and babesiosis. Like other members of the superphylum Alveolata, apicomplexans have regulated exocytosis of specialized secretory organelles, such as the apicomplexan-specific rhoptries and micronemes that are required for host cell invasion. The secretions of another class of organelles, the dense granules and osmiophilic bodies, are proposed to be required for maintenance of the parasitophorous vacuole and host cell egress. Little is known about the osmiophilic bodies and to date only one protein, P377, has been localized to this organelle. In this issue, de Koning-Ward et al. describe the disruption of pfg377 in the virulent human malaria parasite, Plasmodium falciparum, which results in reduced osmiophilic body formation, a marked decrease in female fitness, and dramatically impaired infectivity to mosquitoes. These findings suggest that targeting PFG377 may be a strategy to block parasite transmission.     

Inhibition of LHRH-induced LH and FSH release by gonadotrophin surge-attenuating factor (GnSAF) from human follicular fluid

It has been suggested that in superovulated women the endogenous LH surge is attenuated by a non-steroidal factor, called gonadotrophin surge-attenuating factor (GnSAF), which reduces gonadotrophin secretion in response to LHRH. To determine whether human follicular fluid (hFF) from superovulated women contains GnSAF activity, the secretion of LH and FSH by cultured sheep pituitaries was studied. After charcoal extraction of steroids, hFF was treated by heparin/Sepharose chromatography, which reversibly binds inhibin. The effects of whole hFF and the bound and unbound fractions on basal and LHRH-induced gonadotrophin secretion were then assessed. Steroid-free hFF significantly reduced basal FSH, but not basal LH, secretion, and significantly attenuated the LH and FSH responses to LHRH. The bound (inhibin) fraction significantly decreased both basal and LHRH-induced FSH secretion but did not affect LH release. The unbound fraction had no effect on basal LH or FSH secretion, but significantly attenuated LHRH-induced secretion of both LH and FSH. We conclude that the unbound fraction of hFF from superovulated women contains GnSAF. It has been demonstrated that GnSAF is a non-steroidal factor and its activity is distinct from that of inhibin.     

Sperm transport in the human female reproductive tract in relation to semen analysis characteristics and time of ovulation

Laparoscopic sperm recovery from the pouch of Douglas and tubal fimbriae was performed in 64 infertile couples. Spermatozoa were recovered from 16/35 couples investigated after AIH, and from 13/29 couples post coitum. The method of insemination had no effect on the result, which was positive in 45.3% of all couples, although AIH did result in significantly larger numbers of peritoneal spermatozoa. The number of peritoneal spermatozoa did not show any direct correlation with the number inseminated, but there were reductions along the tract of 5.83 (+/- 1.4 s.d.) orders of magnitude for total sperm count, and 5.52 (+/- 1.21 s.d.) for the number of motile spermatozoa. Only sperm motility had a significant influence on the success of sperm transport; spermatozoa were recovered from patients with sperm densities as low as 3.0 and 3.5 x 10(6)/ml, but with 56 and 44% motile spermatozoa. No influence of cycle day within the range +/- 4 days of ovulation on sperm transport was found. In 45 couples, routine semen analyses were apparently completely normal, but the incidence of sperm recovery was still only 49% (22/45), suggesting that a failure of sperm transport may have been a significant causative factor in their infertility.     

Comparative studies of glutathione reductase and lipoamide dehydrogenase

1. Glutathione reductase and lipoamide dehydrogenase are structurally and mechanistically related flavoenzymes catalyzing various one and two electron transfer reactions between NAD(P)H and substrates with different structures. 2. The two enzymes differ in their coenzyme and functional specificities. Lipoamide dehydrogenase shows higher coenzyme preference while glutathione reductase displays greater functional specificity. 3. Binding preference of the two flavoenzymes for nicotinamide coenzymes is demonstrated by 31P-NMR spectroscopy. 4. The presence of arginines in glutathione reductase which is inactivated by phenyl glyoxal, is likely to be responsible for the NADPH-activity of glutathione reductase. 5. The substrate binding sites of the two enzymes are similar, though their functional details differ. 6. The active-site histidine of glutathione reductase functions primarily as the proton donor during catalysis. While the active-site histidine of lipoamide dehydrogenase stabilizes the thiolate anion intermediate and relays a proton in the catalytic process.