Points to consider in seeking biosafety approval for research, testing, and environmental release of experimental genetically modified biocontrol products during research and development.

Novel genetically modified biological control products (referred to as "GM biocontrol products") are being considered to address a range of complex problems in public health, conservation, and agriculture, including preventing the transmission of vector-borne parasitic and viral diseases as well as the spread of invasive plant and animal species. These interventions involve release of genetically modified organisms (GMOs) into the environment, sometimes with intentional dissemination of the modification within the local population of the targeted species, which presents new challenges and opportunities for regulatory review and decision-making. Practices developed for GMOs, primarily applied to date for GM crops may need to be adapted to accommodate different types of organisms, such as insects, and different technologies, such as gene drive. Developers of new GM biocontrol products would benefit from an early understanding of safety data and information that are likely to be required within the regulatory dossier for regulatory evaluation and decision making. Here a generalizable tool drawing from existing GM crop dossier requirements, forms, and relevant experience is proposed to assist researchers and developers organize and plan their research and trialing. This tool requires considering specifics of each investigational product, their intended use, and country specific requirements at various phases of potential product development, from laboratory research through contained field testing and experimental release into the environment. This may also be helpful to risk assessors and regulators in supporting their systematic and rigorous evaluation of new biocontrol products.

Lentiviruses in gene therapy clinical research.

Gene therapy vectors derived from lentiviruses offer many potentially unique advantages over more conventional retroviral gene delivery systems. Principal amongst these is their ability to provide long-term and stable gene expression and to infect non-dividing cells, such as neurons. However, the use of lentiviral-based vectors in the clinic also raises specific safety and ethical issues. Concerns include the possible generation of replication competent lentiviruses during vector production, mobilisation of the vector by endogenous retroviruses in the genomes of patients, insertional mutagenesis leading to cancer, germline alteration resulting in trans-generational effects and dissemination of new viruses from gene therapy patients. Investigators proposing to conduct this type of research should take due account of the potential risks for interaction of lentiviral gene therapy vectors with other retroviral elements in human subjects, such as Human Immunodeficiency Virus. In addition, strict quality control for replication competent lentiviruses and suitable measurements of lentiviral infectious particle number will be required before these types of viral vector can proceed to the clinic.

Expanded polyglutamines in Caenorhabditis elegans cause axonal abnormalities and severe dysfunction of PLM mechanosensory neurons without cell death.

Huntington's disease (HD) is a dominant neurodegenerative disease caused by polyglutamine (polyQ) expansion in the protein huntingtin (htt). HD pathogenesis appears to involve the production of mutated N-terminal htt, cytoplasmic and nuclear aggregation of htt, and abnormal activity of htt interactor proteins essential to neuronal survival. Before cell death, neuronal dysfunction may be an important step of HD pathogenesis. To explore polyQ-mediated neuronal toxicity, we expressed the first 57 amino acids of human htt containing normal [19 Gln residues (Glns)] and expanded (88 or 128 Glns) polyQ fused to fluorescent marker proteins in the six touch receptor neurons of Caenorhabditis elegans. Expanded polyQ produced touch insensitivity in young adults. Noticeably, only 28 +/- 6% of animals with 128 Glns were touch sensitive in the tail, as mediated by the PLM neurons. Similar perinuclear deposits and faint nuclear accumulation of fusion proteins with 19, 88, and 128 Glns were observed. In contrast, significant deposits and morphological abnormalities in PLM cell axons were observed with expanded polyQ (128 Glns) and partially correlated with touch insensitivity. PLM cell death was not detected in young or old adults. These animals indicate that significant neuronal dysfunction without cell death may be induced by expanded polyQ and may correlate with axonal insults, and not cell body aggregates. These animals also provide a suitable model to perform in vivo suppression of polyQ-mediated neuronal dysfunction.

Integrins: a role for adhesion molecules in olfactory memory.

A gene required for short-term memory in Drosophila, Volado, encodes an alpha integrin and is preferentially expressed in the mushroom bodies of the adult brain. Adhesion molecules of this kind may play a role in olfactory memory by altering the strength of synaptic connections in an experience-dependent manner.

Associative learning disrupted by impaired Gs signaling in Drosophila mushroom bodies.

Disruptions in mushroom body (MB) or central complex (CC) brain structures impair Drosophila associative olfactory learning. Perturbations in adenosine 3',5' monophosphate signaling also disrupt learning. To integrate these observations, expression of a constitutively activated stimulatory heterotrimeric guanosine triphosphate-binding protein alpha subunit (Galphas*) was targeted to these brain structures. The ability to associate odors with electroshock was abolished when Galphas* was targeted to MB, but not CC, structures, whereas sensorimotor responses to these stimuli remained normal. Expression of Galphas* did not affect gross MB morphology, and wild-type Galphas expression did not affect learning. Thus, olfactory learning depends on regulated Gs signaling in Drosophila MBs.

Inducible ternary control of transgene expression and cell ablation in Drosophila.

In Drosophila, P-GAL4 enhancer trap lines can target expression of a cloned gene, under control of a UASGAL element, to any cells of interest. However, additional expression of GAL4 in other cells can produce unwanted lethality or side-effects, particularly when it drives expression of a toxic gene product. To target the toxic gene product ricin A chain specifically to adult neurons, we have superimposed a second layer of regulation on the GAL4 control. We have constructed flies in which an effector gene is separated from UASGAL by a polyadenylation site flanked by two FRT sites in the same orientation. A recombination event between the two FRT sites, catalysed by yeast FLP recombinase, brings the effector gene under control of UASGAL. Consequently, expression of the effector gene is turned on in that cell and its descendants, if they also express GAL4. Recombinase is supplied by heat shock induction of a FLP transgene, allowing both timing and frequency of recombination events to be regulated. Using a lacZ effector (reporter) to test the system, we have generated labelled clones in the embryonic mesoderm and shown that most recombination events occur soon after FLP recombinase is supplied. By substituting the ricin A chain gene for lacZ, we have performed mosaic cell ablations in one GAL4 line that marks the adult giant descending neurons, and in a second which marks mushroom body neurons. In a number of cases we observed loss of one or both the adult giant descending neurons, or of subsets of mushroom body neurons. In association with the mushroom body ablations, we also observed misrouting of surviving axons.

T-cell receptor alpha chain plays a critical role in antigen-specific suppressor cell function.

Antigen-specific suppressor T-cell hybridomas release soluble suppressor factors (TsF) in the supernatant that modulate both in vivo delayed-type hypersensitivity and in vitro plaque-forming cell responses in an antigen-specific manner. To study the relationship between the T-cell receptor (TcR) and TsF, we developed a series of TcR alpha- or TcR beta- expression variants from suppressor T-cell hybridomas that expressed the CD3-TcR alpha/beta complex. We demonstrate that loss of TcR alpha but not TcR beta mRNA was accompanied by the concomitant loss of suppressor bioactivity. Homologous transfection of TcR alpha cDNA into a TcR alpha- beta+ clone reconstituted both CD3-TcR expression and suppressor function. Furthermore, suppressor activity from TcR beta- variants was specifically absorbed by antigen and anti-TcR alpha antibodies, but not by anti-CD3 or anti-TcR beta affinity columns. These data directly establish a role for the TcR alpha chain in suppressor T-cell function and suggest that the TcR alpha chain is part of the antigen-specific TsF molecule.