Biology subjects

Loprieno, M. A.

Publications and source records attributed to Loprieno, M. A..

5 recordsLinked to original sources

bioRxiv2025

Minimization of gene editing off-target effects by tissue restriction of expression

Therapeutic in vivo gene editing with highly specific nucleases has the potential to revolutionize treatment for a wide range of human diseases, including genetic disorders and latent viral infections like herpes simplex virus (HSV). However, challenges regarding specificity, efficiency, delivery, and safety must be addressed before its clinical application. A key concern is the risk of off-target effects, which can cause unintended and potentially harmful genetic changes. We previously developed a curative in vivo gene editing approach to eliminate latent HSV using HSV-specific meganuclease delivered by an AAV vector. In this study, we investigate off-target effects of meganuclease by identifying potential off-target sites through GUIDE-tag analysis and assessing genetic alterations using amplicon deep sequencing in tissues from meganuclease treated mice. Our results show that meganuclease expression driven by a ubiquitous promoter leads to high off-target gene editing in the mouse liver, a non-relevant target tissue. However, restricting the meganuclease expression with a neuron-specific promoter and/or a liver-specific miRNA target sequence efficiently reduces off-target effects in both liver and trigeminal ganglia. These findings suggest that incorporation of regulatory DNA elements for tissue-specific expression in viral vectors can reduce off-target effects and improve the safety of therapeutic in vivo gene editing.

molecular biology↗

bioRxiv2023

Viral gene drive spread during herpes simplex virus 1 infection in mice

Gene drives are genetic modifications designed to propagate efficiently through a population. Most applications rely on homologous recombination during sexual reproduction in diploid organisms such as insects, but we recently developed a gene drive in herpesviruses that relies on co-infection of cells by wild-type and engineered viruses. Here, we developed a viral gene drive against human herpes simplex virus 1 (HSV-1) and showed that it propagated efficiently in vitro and during HSV-1 infection in mice. We observed high levels of co-infection and gene drive-mediated recombination in neuronal tissues during herpes encephalitis as the infection progressed from the site of inoculation to the peripheral and central nervous systems. In addition, we found evidence that a superinfecting gene drive virus could recombine with wild-type viruses during latent infection. These findings indicated that HSV-1 achieves high rates of co-infection and recombination during viral infection, a phenomenon that is currently underappreciated. Overall, this study showed that a viral gene drive could spread in vivo during HSV-1 infection, paving the way toward therapeutic applications.

microbiology↗

bioRxiv2022

AAV-delivered gene editing for latent genital or orofacial herpes simplex virus infection reduces ganglionic viral load and minimizes subsequent viral shedding in mice

Herpes simplex virus (HSV) establishes latency in ganglionic neurons of the peripheral nervous system, from which it can reactivate, causing recurrent disease and possible transmission to a new host. Current anti-HSV therapy does not eliminate latent HSV, and thus is only suppressive rather than curative. We developed a potentially curative approach to latent HSV infection and pathogenesis, based on gene editing using HSV-specific meganucleases delivered by adeno-associated virus (AAV) vectors. Our results demonstrated that a dual meganuclease therapy, composed of two anti-HSV-1 meganucleases delivered by a triple AAV serotype combination (AAV9, AAV-Dj/8, AAV-Rh10), can eliminate up to 97% of latent HSV DNA from ganglia in both ocular and vaginal mouse models of latent HSV infection. Using a novel pharmacological approach to reactivate latent HSV-1 in mice with the bromodomain inhibitor JQ-1, we demonstrated that this reduction in ganglionic viral load leads to a significant reduction of viral shedding from treated vs. control mice, with many treated mice showing no detectable virus shedding. In general, therapy was well tolerated, although dose-ranging studies showed hepatotoxicity at high AAV doses, consistent with previous observations in animals and humans. Also in agreement with previous literature, we observed subtle histological evidence of neuronal injury in some experimental mice, although none of the mice demonstrated observable neurological signs or deficits. These results reinforce the curative potential of gene editing for latent orofacial and genital HSV disease, and provide a framework for additional safety studies before human trials can begin.

neuroscience↗

bioRxiv2022

Liver humanized NSG-PiZ mice support the study of chronic hepatitis B virus infection and antiviral therapies

Hepatitis B virus (HBV) is a pathogen of major public health importance that is largely incurable once a chronic hepatitis B (CHB) infection is established. Only humans and great apes are fully permissive to HBV replication, and this species restriction has impacted HBV research by limiting the utility of small animal models of HBV. To combat the species restriction of HBV and enable more HBV studies in vivo, liver-humanized mouse models have been developed that harbor primary human hepatocytes (PHH) and are fully permissive to HBV infection and replication. Unfortunately, these models can be difficult to establish and are expensive commercially, which has limited their academic use. As an alternative mouse model to study HBV, we evaluated liver-humanized NSG-PiZ mice and showed that they are fully permissive to HBV and can develop CHB. Mice were infected with a precore mutant clinical isolate that has now been serially passaged through 3 generations of mice without loss of fitness. HBV selectively replicates in hCK18+ human hepatocytes within chimeric livers, and HBV+ mice secrete infectious virions and HBsAg into blood, while also harboring covalently closed circular DNA (cccDNA). HBV+ mice remain viremic for at least 169 days, which should enable the study of new curative therapies targeting CHB and respond to antiviral entecavir therapy. The extended duration of viremia is sufficient to enable the study of established and new therapeutic approaches targeting CHB. Furthermore, HBV+ PHH in NSG-PiZ mice can be transduced by the hepatotropic AAV3b and AAV.LK03 vector capsids, which should enable the study of curative gene therapies that target CHB. In summary, our data demonstrates that liver humanized NSG-PiZ mice can be used as a robust and cost-effective alternative to existing CHB models and may enable more academic research labs to study HBV disease pathogenesis and antiviral therapy in a setting that is fully permissive to ongoing replication.

microbiology↗

bioRxiv2020

SARS-CoV-2 ORF6 disrupts nucleocytoplasmic transport through interactions with Rae1 and Nup98

RNA viruses that replicate in the cytoplasm often disrupt nucleocytoplasmic transport to preferentially translate their own transcripts and prevent host antiviral responses. The Sarbecovirus accessory protein ORF6 has previously been shown to be the major inhibitor of interferon production in both SARS-CoV and SARS-CoV-2. SARS-CoV-2 ORF6 was recently shown to co-purify with the host mRNA export factors Rae1 and Nup98. Here, we demonstrate SARS-CoV-2 ORF6 strongly represses protein expression of co-transfected reporter constructs and imprisons host mRNA in the nucleus, which is associated with its ability to co-purify with Rae1 and Nup98. These protein-protein interactions map to the C-terminus of ORF6 and can be abolished by a single amino acid mutation in Met58. Overexpression of Rae1 restores reporter expression in the presence of SARS-CoV-2 ORF6. We further identify an ORF6 mutant containing a 9-amino acid deletion, ORF6 {Delta}22-30, in multiple SARS-CoV-2 clinical isolates that can still downregulate the expression of a co-transfected reporter and interact with Rae1 and Nup98. SARS-CoV ORF6 also interacts with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly co-purifies with Rae1 and Nup98 and results in significantly reduced expression of reporter proteins compared to SARS-CoV ORF6, a potential mechanism for the delayed symptom onset and pre-symptomatic transmission uniquely associated with the SARS-CoV-2 pandemic. ImportanceSARS-CoV-2, the causative agent of COVID-19, is an RNA virus with a large genome that encodes accessory proteins. While these accessory proteins are not required for growth in vitro, they can contribute to the pathogenicity of the virus. One of SARS-CoV-2s accessory proteins, ORF6, was recently shown to co-purify with two host proteins, Rae1 and Nup98, involved in mRNA nuclear export. We demonstrate SARS-CoV-2 ORF6 interaction with these proteins is associated with reduced expression of a reporter protein and accumulation of poly-A mRNA within the nucleus. SARS-CoV ORF6 also shows the same interactions with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly represses reporter expression and co-purifies with Rae1 and Nup98 compared to SARS-CoV ORF6. The ability of SARS-CoV-2 ORF6 to more strongly disrupt nucleocytoplasmic transport than SARS-CoV ORF6 may partially explain critical differences in clinical presentation between the two viruses.

microbiology↗