The unique ability of retroviruses to integrate genes into host genomes is of great value for long-term expression in gene therapy, but only when integrations occur at safe genomic sites. To reap the benefit of using retroviruses without severe detrimental effects, we developed several murine leukemia virus (MLV)-based gammaretroviral vectors with safer integration patterns by perturbing the structure of the integrase via insertion of DNA-binding zinc-finger domains (ZFDs) into an internal position of the enzyme. ZFD insertion significantly reduced the inherent, strong MLV integration preference for genomic regions near transcriptional start sites (TSSs), which are the most dangerous spots. The altered retroviral integration pattern was related to increased formation of residual primer-binding site sequences at the 3′ end of proviruses. Several ZFD insertion mutants showed lower frequencies of integrations into the TSS genome regions when having the residual primer-binding site sequences in the proviruses. Our findings not only can extend the use of retroviruses in biomedical applications, but also provide a glimpse into the mechanisms underlying retroviral integration.
In chromosome 11, 71 out of its 1254
proteins remain functionally
uncharacterized on the basis of their existence evidence (uPE1s) following
the latest version of neXtProt (release 2020-01-17). Because in vivo and in vitro experimental strategies are often time-consuming
and labor-intensive, there is a need for a bioinformatics tool to
predict the function annotation. Here, we used I-TASSER/COFACTOR provided
on the neXtProt web site, which predicts gene ontology (GO) terms
based on the 3D structure of the protein. I-TASSER/COFACTOR predicted
2413 GO terms with a benchmark dataset of the 22 proteins belonging
to PE1 of chromosome 11. In this study, we developed a filtering algorithm
in order to select specific GO terms using the GO map generated by
I-TASSER/COFACTOR. As a result, 187 specific GO terms showed a higher
average precision-recall score at the least cellular component term
compared to 2413 predicted GO terms. Next, we applied 65 proteins
belonging to uPE1s of chromosome 11, and then 409 out of 6684 GO terms
survived, where 103 and 142 GO terms of molecular function and biological
process, respectively, were included. Representatively, the cellular
component GO terms of CCDC90B, C11orf52, and the SMAP were predicted
and validated using the overexpression system into 293T cells and
immunofluorescence staining. We will further study their biological
and molecular functions toward the goal of the neXt-CP50 project as
a part of C-HPP. We shared all results and programs in Github ().
ADNP syndrome, involving the ADNP transcription factor of the SWI/SNF chromatin-remodeling complex, is characterized by developmental delay, intellectual disability, and autism spectrum disorders (ASD). Although Adnp-haploinsufficient (Adnp-HT) mice display various phenotypic deficits, whether these mice display abnormal synaptic functions remain poorly understood. Here, we report synaptic plasticity deficits associated with cognitive inflexibility and CaMKIIα hyperactivity in Adnp-HT mice. These mice show impaired and inflexible contextual learning and memory, additional to social deficits, long after the juvenile-stage decrease of ADNP protein levels to ~10% of the newborn level. The adult Adnp-HT hippocampus shows hyperphosphorylated CaMKIIα and its substrates, including SynGAP1, and excessive long-term potentiation that is normalized by CaMKIIα inhibition. Therefore, Adnp haploinsufficiency in mice leads to cognitive inflexibility involving CaMKIIα hyperphosphorylation and excessive LTP in adults long after its marked expressional decrease in juveniles.
Subtraction of the number of residual vector plasmid molecules from the conventionally measured genomic titer can yield reliable quantification of retroviral and lentiviral vector samples, a prerequisite to advancing the safety of gene therapy applications.
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