There is currently no effective treatment for Friedreich's ataxia (FA), the most common of the hereditary ataxias. The disease is caused by mutations in FRDA that drastically reduce expression levels of the mitochondrial protein frataxin. In FA animal models, a key difficulty is obtaining the precise levels of frataxin expression in the appropriate tissues to provoke pathology without early lethality. To develop strategies to circumvent these problems, conditional frataxin transgenic mice have been generated. We now show that frataxin expression can be eliminated in neurons from these loxP[frda] mice by infection with CRE-expressing herpes simplex virus type 1 (HSV-1) amplicon vectors. We have also achieved in vivo delivery by stereotaxic injection of these CRE-expressing vectors into the brainstem of loxP[frda] mice to generate a localized gene knockout model. These mice develop a behavioral deficit in the rotarod assay detectable after 4 weeks, and when re-injected with HSV-1 amplicon vectors expressing human frataxin complementary DNA (cDNA) exhibit behavioral recovery as early as 4 weeks after the second injection. To the best of our knowledge, this is the first proof of principle of recovery of neurological function by a therapeutic agent aimed at correcting frataxin deficiency.
The bacteriophage f29 replication protein p1 (85 amino acids) is membrane associated in Bacillus subtilis-infected cells. The C-terminal 52 amino acid residues of p1 are suf®cient for assembly into proto®-lament sheet structures. Using chemical cross-linking experiments, we demonstrate here that p1DC43, a C-terminally truncated p1 protein that neither associates with membranes in vivo nor self-interacts in vitro, can interact with the primer terminal protein (TP) in vitro. Like protein p1, plasmid-encoded protein p1DC43 reduces the rate of f29 DNA replication in vivo in a dosage-dependent manner. We also show that truncated p1 proteins that retain the N-terminal 42 amino acids, when present in excess, interfere with the in vitro formation of the TP´dAMP initiation complex in a reaction that depends on the ef®cient formation of a primer TP±f29 DNA polymerase heterodimer. This interference is suppressed by increasing the concentration of either primer TP or f29 DNA polymerase. We propose a model for initiation of in vivo f29 DNA replication in which the viral replisome attaches to a membrane-associated p1-based structure.
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