Mutant sodium channel for tumor therapy.

Viral vectors have been used to deliver a wide range of therapeutic genes to tumors. In this study, a novel tumor therapy was achieved by the delivery of a mammalian brain sodium channel, ASIC2a, carrying a mutation that renders it constitutively open. This channel was delivered to tumor cells using a herpes simplex virus-1/Epstein-Barr virus (HSV/EBV) hybrid amplicon vector in which gene expression was controlled by a tetracycline regulatory system (tet-on) with silencer elements. Upon infection and doxycycline induction of mutant channel expression in tumor cells, the open channel led to amiloride-sensitive sodium influx as assessed by patch clamp recording and sodium imaging in culture. Within hours, tumor cells swelled and died. In addition to cells expressing the mutant channel, adjacent, noninfected cells connected by gap junctions also died. Intratumoral injection of HSV/EBV amplicon vector encoding the mutant sodium channel and systemic administration of doxycycline led to regression of subcutaneous tumors in nude mice as assessed by in vivo bioluminescence imaging. The advantage of this direct mode of tumor therapy is that all types of tumor cells become susceptible and death is rapid with no time for the tumor cells to become resistant.

Targeted integration of functional human ATM cDNA into genome mediated by HSV/AAV hybrid amplicon vector.

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, genome instability, and sensitivity to ionizing radiation (IR). We have previously shown that a herpes simplex virus type 1 (HSV-1) amplicon vector carrying the human ataxia-telangiectasia mutated (ATM) complementary DNA (cDNA) is able to correct aspects of the cellular phenotype of human A-T cells in culture, and is also able to transfer the ATM cDNA to the Atm(-/-) mouse cerebellum. In order to achieve stable gene replacement, we have generated an HSV/adeno-associated virus (AAV) hybrid amplicon vector carrying the expression cassettes for the ATM cDNA [(9.2 kilobases (kb)] and enhanced green fluorescent protein (EGFP), flanked by AAV inverted terminal repeats (ITRs). This hybrid vector, in the presence of AAV Rep proteins, mediates site-specific integration into the AAVS1 site on chromosome 19 in human cells and in Atm(-/-) mice carrying that human locus. The functional activity of the vector-derived ATM was confirmed in vitro and in vivo by ATM autophosphorylation at Ser-1981 after IR. This proof-of-principle study establishes the ability of HSV/AAV hybrid amplicon vectors to mediate functional targeted integration of the ATM cDNA into A-T cells in culture and in Atm(-/-) mice in vivo, thus laying a foundation for possible gene therapy approaches in the treatment of A-T patients.

Metabolic biotinylation of cell surface receptors for in vivo imaging.

We have developed a versatile, potent technique for imaging cells in culture and in vivo by expressing a metabolically biotinylated cell-surface receptor and visualizing it with labeled streptavidin moieties. The recombinant reporter protein, which incorporates a biotin acceptor peptide (BAP) between an N-terminal signal sequence and a transmembrane domain, (BAP-TM) was efficiently biotinylated by endogenous biotin ligase in mammalian cells with the biotin displayed on the cell surface. Tumors expressing the BAP-TM have high sensitivity for magnetic resonance and fluorescence tomographic imaging in vivo after intravascular injection of streptavidin conjugated to magnetic nanoparticles or fluorochromes, respectively. Moreover, streptavidin-horseradish peroxidase conjugates in conjunction with a peroxidase-sensitive gadolinium agent further increased and prolonged the magnetic resonance signal. This BAP-TM allows noninvasive real-time imaging of any cell type transduced to express this reporter protein in culture or in vivo.

Antipsychotic medication during pregnancy and lactation in women with schizophrenia: evaluating the risk.

OBJECTIVE: To review studies investigating the following: whether exposing developing infants to antipsychotic medication during pregnancy and lactation is associated with increased risks of teratogenic, neonatal, and long-term neurobehavioural sequelae; whether schizophrenia itself affects pregnancy outcome; and whether the course of schizophrenia symptoms is altered by pregnancy and lactation. METHOD: We summarize the results from articles identified via a MedLine search for the period January 1, 1966, to December 1, 2001. RESULTS: Women with schizophrenia are at increased risk for poor obstetrical outcomes, including preterm delivery, low birth weight, and neonates who are small for their gestational age. A lack of information in the literature makes it difficult to comment on the relative risk of exposing developing infants to atypical antipsychotics. However, typical antipsychotics appear to carry an increased risk of congenital malformations when the fetus is exposed to phenothiazines during weeks 4 to 10 of gestation. Lack of information also precludes an understanding of whether changes associated with pregnancy and lactation significantly alter the course of schizophrenia symptoms. CONCLUSION: Research is needed so that physicians may more accurately inform women about the relative risks of using antipsychotic medications during pregnancy and lactation. Increased knowledge about the risks of medication exposure will allow clinicians to limit treatment to situations in which the risk of untreated maternal illness outweighs the risk of exposing a developing infant to medications.