The Molecular Virology and Neurogenetics Section focuses on the development of lentiviral vectors for the delivery of transgenes to motoneurons by either retrograde transport after intramuscular injection or by direct injection into the spinal cord. Depending on the efficacy of these routes of transgene delivery, select animal models for motoneuron diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy will be addressed. In addition, these vectors will be used to study basic motoneuron function in locomotion. Other targets are cells in epileptic foci in a rat amygdala model for focal epilepsy or hyperactive neurons in chronic pain. Transgene expression in some of these target cells shares vector elements such as promoters and transgenes, targeted delivery routes and vector isolation procedures, which are at the center of current studies. Two promoter elements were identified, which may allow injury- or activity-induced expression of neuroprotective and/or neuronal silencing transgenes. These elements were initially identified in a cytomegalovirus immediate/early promoter (CMV), which is constitutively active at high level in astrocytes, but shows regulated expression in neurons. The promoter elements thought to be involved were identified and separately fused to a minimal constitutive, neuron-specific CaMKII-alpha promoter. These additions to the promoter now combine neuron-specificity with inducibility of promoter activity by either injury-associated proteins such as TNF-alpha or by depolarization and Ca2+ influx. In addition, a stronger neuron-specific human synapsin 1 promoter was cloned, which was active in more types of neurons and could also be combined with these inducible elements. For neuronal silencing, an inwardly rectifying potassium channel Kir2.1 was expressed under control of the CMV promoter. The results indicated that Kir2.1 may autoregulate its own expression from a CMV promoter and silence neurons. Studies to identify elements within the promoter and within the Kir2.1 protein involved in this postulated autoregulation are in progress. These elements may potentially be directed towards neuronal silencing i.e. during chronic pain or to study mechanisms of coordinated neuronal activity during locomotion. A second candidate neuronal silencer gene was synthesized, which encodes the neuropeptide galanin fused to the fibronectin signal peptide to permit efficient secretion from cells. Unlike Kir2.1, its activity will not be limited to the transduced cells. Secretion expands its sphere of influence presumably via the remote activation of G-protein activated potassium channels in neighboring cells. Efficacy of this approach will be evaluated in a kindled rat model of focal epilepsy. For transgene delivery, we are currently focusing on motoneurons and on potential retrograde transport of lentiviral vectors by pseudotype vector formation using rabies virus glycoproteins that had been cloned from nine different rabies virus strains, which differ in neurotropism, neuroinvasiveness and neuropathogenesis. Several high titer pseudotype lentiviral vectors have been isolated. Intramuscular injections of some of these pseudotype particles resulted in retrograde transport and reporter gene expression in a few motoneurons in the spinal cord. High titer vector preparations have now been isolated. They will be tested by intramuscular and direct spinal cord injections into mice. Several vector genomes have already been assembled to be packaged into these targeted lentiviral vectors. After identifying the most efficient glycoprotein for retrograde transport, genes affected by several motoneuron diseases and by Alzheimer?s disease will either be directly replaced, like SMN1 in spinal muscular atrophy, or expressed to overcome dysfunctional neuronal activities including hyperphosphorylation associated with Alzheimer?s disease and some types of amyotroppphic lateral sclerosis that result in apoptosis. In addition, Kir2.1 will be delivered and expressed in motoneurons and in neurons involved for example in chronic pain.
| Mentis, George Z; Gravell, Maneth; Hamilton, Rebecca et al. (2006) Transduction of motor neurons and muscle fibers by intramuscular injection of HIV-1-based vectors pseudotyped with select rabies virus glycoproteins. J Neurosci Methods 157:208-17 |
| Ye, Zhiping; Harmison, George G; Ragheb, Jack A et al. (2005) Targeted infection of HIV-1 Env expressing cells by HIV(CD4/CXCR4) vectors reveals a potential new rationale for HIV-1 mediated down-modulation of CD4. Retrovirology 2:80 |
| Zheng, Ya-Li; Kesavapany, Sashi; Gravell, Maneth et al. (2005) A Cdk5 inhibitory peptide reduces tau hyperphosphorylation and apoptosis in neurons. EMBO J 24:209-20 |
