Vectors derived from human immunodeficiency virus (HIV) are highly efficient vehicles for in vivo gene delivery. However, their biosafety is of major concern. Here we exploit the complexity of the HIV genome to provide lentivirus vectors with novel biosafety features. In addition to the structural genes, HIV contains two regulatory genes, tat and rev, that are essential for HIV replication, and four accessory genes that encode critical virulence factors. We previously reported that the HIV type 1 accessory open reading frames are dispensable for efficient gene transduction by a lentivirus vector. We now demonstrate that the requirement for the tat gene can be offset by placing constitutive promoters upstream of the vector transcript. Vectors generated from constructs containing such a chimeric long terminal repeat (LTR) transduced neurons in vivo at very high efficiency, whether or not they were produced in the presence of Tat. When the rev gene was also deleted from the packaging construct, expression of gag and pol was strictly dependent on Rev complementation in trans. By the combined use of a separate nonoverlapping Rev expression plasmid and a 5′ LTR chimeric transfer construct, we achieved optimal yields of vector of high transducing efficiency (up to 107transducing units [TU]/ml and 104 TU/ng of p24). This third-generation lentivirus vector uses only a fractional set of HIV genes: gag, pol, and rev. Moreover, the HIV-derived constructs, and any recombinant between them, are contingent on upstream elements and trans complementation for expression and thus are nonfunctional outside of the vector producer cells. This split-genome, conditional packaging system is based on existing viral sequences and acts as a built-in device against the generation of productive recombinants. While the actual biosafety of the vector will ultimately be proven in vivo, the improved design presented here should facilitate testing of lentivirus vectors.
Recombinant adeno-associated virus 2 (rAAV2) has been shown to deliver genes to neurons effectively in the brain, retina, and spinal cord. The characterization of new AAV serotypes has revealed that they have different patterns of transduction in diverse tissues. We have investigated the tropism and transduction frequency in the central nervous system (CNS) of three different rAAV vector serotypes. The vectors contained AAV2 terminal repeats flanking a green fluorescent protein expression cassette under the control of the synthetic CBA promoter, in AAV1, AAV2, or AAV5 capsids, producing the pseudotypes rAAV2/1, rAAV2/2, and rAAV2/5. Rats were injected with rAAV2/1, rAAV2/2, or rAAV2/5 into selected regions of the CNS, including the hippocampus (HPC), substantia nigra (SN), striatum, globus pallidus, and spinal cord. In all regions injected, the three vectors transduced neurons almost exclusively. All three vectors transduced the SN pars compacta with high efficiency, but rAAV2/1 and rAAV2/5 also transduced the pars reticulata. Moreover, rAAV2/1 showed widespread distribution throughout the entire midbrain. In the HPC, rAAV2/1 and rAAV2/5 targeted the pyramidal cell layers in the CA1-CA3 regions, whereas AAV2/2 primarily transduced the hilar region of the dentate gyrus. In general, rAAV2/1 and rAAV2/5 exhibited higher transduction frequencies than rAAV2/2 in all regions injected, although the differences were marginal in some regions. Retrograde transport of rAAV1 and rAAV5 was also observed in particular CNS areas. These results suggest that vectors based on distinct AAV serotypes can be chosen for specific applications in the nervous system.
Recombinant adeno-associated viral vectors display efficient tropism for transduction of the dopamine neurons of the substantia nigra. Taking advantage of this unique property of recombinant adeno-associated viral vectors, we expressed wild-type and A53T mutated human alpha-synuclein in the nigrostriatal dopamine neurons of adult rats for up to 6 months. Cellular and axonal pathology, including alpha-synuclein-positive cytoplasmic inclusions and swollen, dystrophic neurites similar to those seen in brains from patients with Parkinson's disease, developed progressively over time. These pathological alterations occurred preferentially in the nigral dopamine neurons and were not observed in other nondopaminergic neurons transduced by the same vectors. The degenerative changes were accompanied by a loss of 30-80% of the nigral dopamine neurons, a 40-50% reduction of striatal dopamine, and tyrosine hydroxylase levels that was fully developed by 8 weeks. Significant motor impairment developed in those animals in which dopamine neuron cell loss exceeded a critical threshold of 50-60%. At 6 months, signs of cell body and axonal pathology had subsided, suggesting that the surviving neurons had recovered from the initial insult, despite the fact that alpha-synuclein expression was maintained at a high level. These results show that nigral dopamine neurons are selectively vulnerable to high levels of either wild-type or mutant alpha-synuclein, pointing to a key role for alpha-synuclein in the pathogenesis of Parkinson's disease. Targeted overexpression of alpha-synuclein in the nigrostriatal system may provide a new animal model of Parkinson's disease that reproduces some of the cardinal pathological, neurochemical, and behavioral features of the human disease.
In vivo transduction of nondividing cells by human immunodeficiency virus type 1 (HIV-1)-based vectors results in transgene expression that is stable over several months. However, the use of HIV-1 vectors raises concerns about their safety. Here we describe a self-inactivating HIV-1 vector with a 400-nucleotide deletion in the 3′ long terminal repeat (LTR). The deletion, which includes the TATA box, abolished the LTR promoter activity but did not affect vector titers or transgene expression in vitro. The self-inactivating vector transduced neurons in vivo as efficiently as a vector with full-length LTRs. The inactivation design achieved in this work improves significantly the biosafety of HIV-derived vectors, as it reduces the likelihood that replication-competent retroviruses will originate in the vector producer and target cells, and hampers recombination with wild-type HIV in an infected host. Moreover, it improves the potential performance of the vector by removing LTR sequences previously associated with transcriptional interference and suppression in vivo and by allowing the construction of more-stringent tissue-specific or regulatable vectors.
EEG and single-unit techniques have been used to study the EEG correlates of cellular firing in the neocortex, n. reticularis (RT) and "specific" thalamic nuclei, and the cholinergic forebrain area (nucleus basalis, NB). Neuronal firing was related to the ongoing behavior of the rat. In addition, using a 16-channel neocortical recording/mapping system, we studied the effects of ibotenic acid lesion of NB, RT, and other thalamic nuclei on the patterns and spatial distribution of neocortical electrical activity. The majority of neurons in neocortex, NB, and RT increased their firing rates during walking, as compared to during immobility, with concurrent decrease of delta power in the neocortical EEG. During immobility, high-voltage spindles (HVS; greater than 1 mV) were occasionally recorded from the neocortex. Depth profiles of HVS and slow delta waves were different in the neocortex. Neocortical cells decreased their discharge frequency during the positive portion of delta waves recorded in layers V and VI. All cells in the neocortex and specific thalamic nuclei fired rhythmically and phase-locked to the spike component of HVS. RT neurons showed an opposite phase relationship and fired mainly during the wave component of HVS. Half of the NB neurons also showed phasic modulation with HVS. Circumscribed lesion of RT and extensive damage of other thalamic regions, including the intralaminar nuclei, suppressed HVS but had no effect on the neocortical EEG correlates of behavior. In sharp contrast, damage to the NB resulted in a dramatic increase of slow delta waves on the side of the lesion, mimicking the effect of scopolamine administration. We suggest that the NB plays a key role in neocortical arousal by directly activating the neocortex and by suppressing the rhythm generation in the RT-thalamocortical circuitry. We further suggest that the NB system may serve as a structural basis for the concept of the generalized ascending activation of Moruzzi and Magoun (1949).
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