CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics

The development of personalized medicine for genetic diseases requires preclinical testing in the appropriate animal models. GNAO1 encephalopathy is a severe neurodevelopmental disorder caused by heterozygous de novo mutations in the GNAO1 gene. GNAO1 c.607 G>A is one of the most common pathogenic variants, and the mutant protein Gαo-G203R likely adversely affects neuronal signaling. As an innovative approach, sequence-specific RNA-based therapeutics such as antisense oligonucleotides or effectors of RNA interference are potentially applicable for selective suppression of the mutant GNAO1 transcript. While in vitro validation can be performed in patient-derived cells, a humanized mouse model to rule out the safety of RNA therapeutics is currently lacking. In the present work, we employed CRISPR/Cas9 technology to introduce a single-base substitution into exon 6 of the Gnao1 to replace the murine Gly203-coding triplet (GGG) with the codon used in the human gene (GGA). We verified that genome-editing did not interfere with the Gnao1 mRNA or Gαo protein synthesis and did not alter localization of the protein in the brain structures. The analysis of blastocysts revealed the off-target activity of the CRISPR/Cas9 complexes; however, no modifications of the predicted off-target sites were detected in the founder mouse. Histological staining confirmed the absence of abnormal changes in the brain of genome-edited mice. The created mouse model with the “humanized” fragment of the endogenous Gnao1 is suitable to rule out unintended targeting of the wild-type allele by RNA therapeutics directed at lowering GNAO1 c.607 G>A transcripts

Adeno-Associated Viruses for Modeling Neurological Diseases in Animals: Achievements and Prospects

Adeno-associated virus (AAV) vectors have become an attractive tool for efficient gene transfer into animal tissues. Extensively studied as the vehicles for therapeutic constructs in gene therapy, AAVs are also applied for creating animal models of human genetic disorders. Neurological disorders are challenging to model in laboratory animals by transgenesis or genome editing, at least partially due to the embryonic lethality and the timing of the disease onset. Therefore, gene transfer with AAV vectors provides a more flexible option for simulating genetic neurological disorders. Indeed, the design of the AAV expression construct allows the reproduction of various disease-causing mutations, and also drives neuron-specific expression. The natural and newly created AAV serotypes combined with various delivery routes enable differentially targeting neuronal cell types and brain areas in vivo. Moreover, the same viral vector can be used to reproduce the main features of the disorder in mice, rats, and large laboratory animals such as non-human primates. The current review demonstrates the general principles for the development and use of AAVs in modeling neurological diseases. The latest achievements in AAV-mediated modeling of the common (e.g., Alzheimer’s disease, Parkinson’s disease, ataxias, etc.) and ultra-rare disorders affecting the central nervous system are described. The use of AAVs to create multiple animal models of neurological disorders opens opportunities for studying their mechanisms, understanding the main pathological features, and testing therapeutic approaches

Mengovirus-Induced Rearrangement of the Nuclear Pore Complex: Hijacking Cellular Phosphorylation Machinery▿

Representatives of several picornavirus genera have been shown previously to significantly enhance noncontrollable bidirectional exchange of proteins between nuclei and cytoplasm. In enteroviruses and rhinoviruses, enhanced permeabilization of the nuclear pores appears to be primarily due to proteolytic degradation of some nucleoporins (protein components of the pore), whereas this effect in cardiovirus-infected cells is triggered by the leader (L) protein, devoid of any enzymatic activities. Here, we present evidence that expression of L alone was sufficient to cause permeabilization of the nuclear envelope in HeLa cells. In contrast to poliovirus, mengovirus infection of these cells did not elicit loss of nucleoporins Nup62 and Nup153 from the nuclear pore complex. Instead, nuclear envelope permeabilization was accompanied by hyperphosphorylation of Nup62 in cells infected with wild-type mengovirus, whereas both of these alterations were suppressed in L-deficient virus mutants. Since phosphorylation of Nup62 (although less prominent) did accompany permeabilization of the nuclear envelope prior to its mitotic disassembly in uninfected cells, we hypothesize that cardiovirus L alters the nucleocytoplasmic traffic by hijacking some components of the normal cell division machinery. The variability and biological significance of picornaviral interactions with the nucleocytoplasmic transport in the infected cells are discussed

Posttranscriptional Regulation of HIV-1 Gene Expression during Replication and Reactivation from Latency by Nuclear Matrix Protein MATR3.

Posttranscriptional regulation of HIV-1 replication is finely controlled by viral and host factors. Among the former, Rev controls the export of partially spliced and unspliced viral RNAs from the nucleus and their translation in the cytoplasm or incorporation into new virions as genomic viral RNA. To investigate the functional role of the Rev cofactor MATR3 in the context of HIV infection, we modulated its expression in Jurkat cells and primary peripheral blood lymphocytes (PBLs). We confirmed that MATR3 is a positive regulator of HIV-1 acting at a posttranscriptional level. By applying the same approach to J-lat cells, a well-established model for the study of HIV-1 latency, we observed that MATR3 depletion did not affect transcriptional reactivation of the integrated provirus, but caused a reduction of Gag production. Following these observations, we hypothesized that MATR3 could be involved in the establishment of HIV-1 posttranscriptional latency. Indeed, mechanisms acting at the posttranscriptional level have been greatly overlooked in favor of transcriptional pathways. MATR3 was almost undetectable in resting PBLs, but could be promptly upregulated upon cellular stimulation with PHA. However, HIV latency-reversing agents were poor inducers of MATR3 levels, providing a rationale for their inability to fully reactivate the virus. These data have been confirmed ex vivo in cells derived from patients under suppressive ART. Finally, in the context of MATR3-depleted J-lat cells, impaired reactivation by SAHA could be fully rescued by MATR3 reconstitution, demonstrating a direct role of MATR3 in the posttranscriptional regulation of HIV-1 latency.IMPORTANCE The life cycle of HIV-1 requires integration of a DNA copy into the genome of the host cell. Transcription of the viral genes generates RNAs that are exported to the cytoplasm with the contribution of viral and cellular factors to get translated or incorporated in the newly synthesized virions. It has been observed that highly effective antiretroviral therapy, which is able to reduce circulating virus to undetectable levels, cannot fully eradicate the virus from cellular reservoirs that harbor a transcriptionally latent provirus. Thus, persistence of latently infected cells is the major barrier to a cure for HIV-1 infection. In order to purge these reservoirs of latently infected cells, it has been proposed to activate transcription to stimulate the virus to complete its life cycle. This strategy is believed to unmask these reservoirs, making them vulnerable to the immune system. However, limited successes of this approach may indicate additional posttranscriptional restrictions that need to be overcome for full virus reactivation. In this work we identify the cellular protein MATR3 as an essential cofactor of viral RNA processing. Reactivation of HIV-1 transcription per se is not sufficient to allow completion of a full life cycle of the virus if MATR3 is depleted. Furthermore, MATR3 is poorly expressed in quiescent CD4+ T lymphocytes that are the major reservoir of latent HIV-1. Cells derived from aviremic HIV-1 patients under antiretroviral therapy didn't express MATR3, and most importantly, latency-reversing agents proposed for the rescue of latent provirus were ineffective for MATR3 upregulation. To conclude, our work identifies a cellular factor required for full HIV-1 reactivation and points to the revision of the current strategies for purging viral reservoirs that focus only on transcription.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Nucleocytoplasmic Traffic Disorder Induced by Cardioviruses

Some picornaviruses, for example, poliovirus, increase bidirectional permeability of the nuclear envelope and suppress active nucleocytoplasmic transport. These activities require the viral protease 2A(pro). Here, we studied nucleocytoplasmic traffic in cells infected with encephalomyocarditis virus (EMCV; a cardiovirus), which lacks the poliovirus 2A(pro)-related protein. EMCV similarly enhanced bidirectional nucleocytoplasmic traffic. By using the fluorescent “Timer” protein, which contains a nuclear localization signal, we showed that the cytoplasmic accumulation of nuclear proteins in infected cells was largely due to the nuclear efflux of “old” proteins rather than impaired active nuclear import of newly synthesized molecules. The nuclear envelope of digitonin-treated EMCV-infected cells permitted rapid efflux of a nuclear marker protein. Inhibitors of poliovirus 2A(pro) did not prevent the EMCV-induced efflux. Extracts from EMCV-infected cells and products of in vitro translation of viral RNAs contained an activity increasing permeability of the nuclear envelope of uninfected cells. This activity depended on the expression of the viral leader protein. Mutations disrupting the zinc finger motif of this protein abolished its efflux-inducing ability. Inactivation of the L protein phosphorylation site (Thr47→Ala) resulted in a delayed efflux, while a phosphorylation-mimicking (Thr47→Asp) replacement did not significantly impair the efflux-inducing ability. Such activity of extracts from EMCV-infected cells was suppressed by the protein kinase inhibitor staurosporine. As evidenced by electron microscopy, cardiovirus infection resulted in alteration of the nuclear pores, but it did not trigger degradation of the nucleoporins known to be degraded in the poliovirus-infected cells. Thus, two groups of picornaviruses, enteroviruses and cardioviruses, similarly alter the nucleocytoplasmic traffic but achieve this by strikingly different mechanisms