Pramyxovirus vectors encoding antibody and utilization thereof
A paramyxovirus and vector technology, which is applied in the field of paramyxovirus vectors to achieve extremely high application value and high application value.
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Embodiment 1
[0138] [Example 1] Construction of the SeV vector carrying the Fab gene
[0139] The application of SeV vectors to spinal cord injury sites exemplifies therapeutic vectors aimed at inhibiting axonal elongation inhibitors such as NOGO. Since it is known that IN-1 (mouse IgMκ type) is a neutralizing antibody against NOGO (Brosamle, C. et al., J. Neurosci. 20 (21), 8061-8068 (2000) etc.), the construct carrying IN-1 The spreading SeV vector of -1 gene. SeV vectors deficient in the F-gene (transmissibility deficient) were also constructed.
[0140] 1) total gene synthesis
[0141] In order to construct a SeV vector carrying the Fab (H and L chain) genes of IN-1, total synthesis of the Fab genes of IN-1 was performed. According to the base sequence of the single-chain Fab fragment of IN-1 (Accession No. Y08011; Bandtlow, C.et al., Eur.J.Biochem.241(2)468-475(1996)), the sequence was designed such that the His-tag was removed, both ends contained NotI recognition sites, and t...
Embodiment 2
[0160] [Example 2] In vitro functional evaluation of SeV carrying the IN-1 gene
[0161] IN-1 is known as a neutralizing antibody against the axonal elongation inhibitor NOGO (Chen, M.S. et al., Nature 403, 434-439 (2000)). Therefore, in order to functionally evaluate SeV carrying the Fab gene of IN-1, it is necessary to observe the promotion of axonal elongation under conditions that inhibit axonal elongation, that is, in the presence of an axonal elongation inhibitor. active. The spinal cord aspirate containing inhibitors was called q-pool, which was prepared according to the method of Spillmann et al. (Spillmann, A.A. et al., J. Biol. Chem. 273, 19283-19293 (1998)). The spinal cord was extracted from three adult rats to obtain 1.5mgq-pool. IN-1 activity was determined according to the methods of Chen and Spillmann et al. (Chen, M.S. et al., Nature 403, 434-439 (2000); Spillmann, A.A. et al., J. Biol. Chem. 273, 19283-19293 (1998)) for evaluation. Two methods were used t...
Embodiment 3
[0164] [Example 3] In vivo evaluation system for evaluating vector expression persistence and expression after repeated administration
[0165] In order to evaluate the persistence of vector expression and the possibility of repeated administration, it is important to establish a more effective and reliable in vivo evaluation system. This example discloses an evaluation system using a newly developed intraauricular drug delivery in mice. The results showed that the spreading SeV vector carrying the GFP gene (SeV18+GFP: 5×10 6 GFP-CIU / 5μl) or F gene-deficient SeV vector (SeV18+GFP / ΔF: 5×10 6 GFP-CIU / 5 μl) was intraauricularly administered to mice, and it was possible to noninvasively observe the fluorescence of GFP protein expressed in infected cells from the outside ( FIG. 9 ). This evaluation system is non-invasive and can observe the expression of SeV vector-derived protein (GFP) over time using the same individual, so this system is very suitable for evaluating the persis...
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