AAV vectors targeted to oligodendrocytes
A technology of oligodendrocytes and vectors, applied in the direction of vectors, cells modified by introducing foreign genetic materials, using vectors to introduce foreign genetic materials, etc.
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Embodiment 1
[0252] Discovery and characterization of BNP61 AAV clone
[0253] The AAV capsid library shuffled with mutant DNA was injected intravenously into a rat model of Parkinson's disease, and cells were isolated from the caudate nucleus after 3 days. Using PCR rescue, a single clone (BNP61) appeared. Additional shuffling and selection resulted in identical clones. Such as figure 1 As shown, this clone is a chimera of several AAV serotypes.
[0254] The BNP61 clone was directly infused into the rat brain, resulting in an amazing pattern of cell transduction. So far, when gene expression is driven by a constitutive promoter, almost all different AAV serotypes and chimeras have shown greater neuronal effects. 95% tropism. The significant difference is that BNP61 showed> 95% tropism, without evidence of astrocyte or microglia transduction and very little neuronal transduction ( figure 2 A-2C ). figure 2A It shows GFP-positive oligodendrocytes in the rat caudate nucleus one week aft...
Embodiment 2
[0258] BNP61 crosses the affected blood-brain barrier
[0259] After the first round of selection, the BNP61 clone was packaged with GFP and a recombinant virus was produced. Then, 2 weeks after 6-OHDA treatment, press 8×10 11 The recombinant virus was administered intravenously at a dose of one vector genome / kg. One month later, the rats were sacrificed and the brains were sliced. In rats 2 weeks after these treatments, a large number of gene expressions were found in oligodendrocytes and some neurons in the striatum treated with 6-OHDA ( Figure 5 ), and no gene expression was found in the contralateral striatum, in the cortex or the syringe tract of the distal brain structure. Note: lack of colocalization of NeuN with many oligodendrocytes surrounding individual NeuN-positive neurons ( Figure 5 ). Therefore, it appears that after intravenous administration, this new AAV clone exhibits the ability to cross the 6-OHDA-damaged blood-brain barrier instead of the complete blo...
Embodiment 3
[0261] Generation of additional targeted clones of oligodendrocytes
[0262] Using the same AAV DNA capsid shuffling and in vivo directed evolution process described in Example 1, two additional oligodendrocyte targeting clones BNP62 and BNP63 were identified. Such as figure 1 As shown and similar to BNP61, these clones are chimeras of several AAV serotypes. See the amino acid sequence identity between these 3 clones Table 2 . All 3 clones are more than 95% identical to each other at the amino acid level.
[0263] Table 2: Sequence identity between clones
[0264] BNP61 BNP62 BNP63 BNP61 100% 96% 97% BNP62 96% 100% 96% BNP63 97% 96% 100%
[0265] Image 6 The BNP63 clone was shown to transduce oligodendrocytes in the rat piriform cortex. BNP63 transduced cells (green) are not co-localized with the cell markers of astrocytes (GFAP, red). In addition, the transduced cells showed clear oligodendrocyte morphology.
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