Extracellular Vesicles Loaded with Long Antisense RNAs Repress Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Long antisense RNAs (asRNAs) have been observed to repress HIV and other virus expression in a manner that is refractory to viral evolution. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) disease, has a distinct ability to evolve resistance around antibody targeting, as was evident from the emergence of various SARS-CoV-2 spike antibody variants. Importantly, the effectiveness of current antivirals is waning due to the rapid emergence of new variants of concern, more recently the omicron variant.

An intranasally delivered ultra-conserved siRNA prophylactically represses SARS-CoV-2 infection in the lung and nasal cavity.

There remains a striking overall mortality burden of COVID-19 worldwide. Given the waning effectiveness of current SARS-CoV-2 antivirals due to the rapid emergence of new variants of concern (VOC), we employed a direct-acting molecular therapy approach using gene silencing RNA interference (RNAi) technology. In this study, we developed and screened several ultra-conserved small-interfering RNAs (siRNAs) before selecting one potent siRNA candidate for pre-clinical in vivo testing.

Evaluation of the Elements of Short Hairpin RNAs in Developing shRNA-Containing CAR T Cells.

Short hairpin RNAs (shRNAs) have emerged as a powerful tool for gene knockdown in various cellular systems, including chimeric antigen receptor (CAR) T cells. However, the elements of shRNAs that are crucial for their efficacy in developing shRNA-containing CAR T cells remain unclear. In this study, we evaluated the impact of different shRNA elements, including promoter strength, orientation, multiple shRNAs, self-targeting, and sense and antisense sequence composition on the knockdown efficiency of the target gene in CAR T cells.

Prophylactic intranasal administration of lipid nanoparticle formulated siRNAs reduce SARS-CoV-2 and RSV lung infection.

RNA interference (RNAi) is an emerging and promising therapy for a wide range of respiratory viral infections. This highly specific suppression can be achieved by the introduction of short-interfering RNA (siRNA) into mammalian systems, resulting in the effective reduction of viral load. Unfortunately, this has been hindered by the lack of a good delivery system, especially via the intranasal (IN) route.

Extracellular vesicles: The next generation in gene therapy delivery.

Extracellular vesicles (EVs) are esteemed as a promising delivery vehicle for various genetic therapeutics. They are relatively inert, non-immunogenic, biodegradable, and biocompatible. At least in rodents, they can even transit challenging bodily hurdles such as the blood-brain barrier.

Targeted zinc-finger repressors to the oncogenic HBZ gene inhibit adult T-cell leukemia (ATL) proliferation.

Human T-lymphotropic virus type I (HTLV-I) infects CD4+ T-cells resulting in a latent, life-long infection in patients. Crosstalk between oncogenic viral factors results in the transformation of the host cell into an aggressive cancer, adult T-cell leukemia/lymphoma (ATL). ATL has a poor prognosis with no currently available effective treatments, urging the development of novel therapeutic strategies.

Pre-clinical data supporting immunotherapy for HIV using CMV-HIV-specific CAR T cells with CMV vaccine.

T cells engineered to express HIV-specific chimeric antigen receptors (CARs) represent a promising strategy to clear HIV-infected cells, but to date have not achieved clinical benefits. A likely hurdle is the limited T cell activation and persistence when HIV antigenemia is low, particularly during antiretroviral therapy (ART). To overcome this issue, we propose to use a cytomegalovirus (CMV) vaccine to stimulate CMV-specific T cells that express CARs directed against the HIV-1 envelope protein gp120.

Harnessing Rift Valley fever virus NSs gene for cancer gene therapy.

One of the greatest challenges in the treatment of cancer is tumor heterogeneity which results in differential responses to chemotherapy and drugs that work through a single pathway. A therapeutic agent that targets cancer cells for death through multiple mechanisms could be advantageous as a broad inhibitor for many types of cancers and the heterogeneous alterations they possess. Several viral proteins have been exploited for antiproliferative and apoptotic effect in cancer cells by disrupting critical survival pathways.

Nuclear microRNA-466c regulates Vegfa expression in response to hypoxia.

MicroRNAs are well characterized in their role in silencing gene expression by targeting 3´-UTR of mRNAs in cytoplasm. However, recent studies have shown that miRNAs have a role in the regulation of genes in the nucleus, where they are abundantly located. We show here that in mouse endothelial cell line (C166), nuclear microRNA miR-466c participates in the regulation of vascular endothelial growth factor a (Vegfa) gene expression in hypoxia.

RNAi to treat SARS-CoV-2-variant proofing the next generation of therapies.

There is an urgent need to bring new antivirals to SARS-CoV-2 to the market. Indeed, in the last 3 months, we have seen at least two new antivirals approved, molnupiravir and paxlovid. Both are older established antivirals that show some efficacy against SARS-CoV-2.

Engineered extracellular vesicles directed to the spike protein inhibit SARS-CoV-2.

SARS-CoV-2 (CoV-2) viral infection results in COVID-19 disease, which has caused significant morbidity and mortality worldwide. A vaccine is crucial to curtail the spread of SARS-CoV-2, while therapeutics will be required to treat ongoing and reemerging infections of SARS-CoV-2 and COVID-19 disease. There are currently no commercially available effective anti-viral therapies for COVID-19, urging the development of novel modalities.

Exosome-mediated stable epigenetic repression of HIV-1.

Human Immunodeficiency Virus (HIV-1) produces a persistent latent infection. Control of HIV-1 using combination antiretroviral therapy (cART) comes at the cost of life-shortening side effects and development of drug-resistant HIV-1. An ideal and safer therapy should be deliverable in vivo and target the stable epigenetic repression of the virus, inducing a stable "block and lock" of virus expression.

Pseudogenes: A Novel Source of Trans-Acting Antisense RNAs.

Several recent studies support a functional role for pseudogenes, a copy of a parent gene that has lost protein-coding potential, which was for a long time thought to represent only "junk" DNA. Several hundreds of pseudogenes have now been reported as transcribed RNAs in a large variety of tissues and tumor types. Most studies have focused on pseudogenes expressed in sense direction, relative to their protein-coding gene counterpart, but some reports suggest that pseudogenes can be also transcribed as antisense RNAs (asRNAs).

A SARS-CoV-2 targeted siRNA-nanoparticle therapy for COVID-19.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans. Despite several emerging vaccines, there remains no verifiable therapeutic targeted specifically to the virus. Here we present a highly effective small interfering RNA (siRNA) therapeutic against SARS-CoV-2 infection using a novel lipid nanoparticle (LNP) delivery system.

Enhanced target cell specificity and uptake of lipid nanoparticles using RNA aptamers and peptides.

Lipid nanoparticles (LNPs) constitute a facile and scalable approach for delivery of payloads to human cells. LNPs are relatively immunologically inert and can be produced in a cost effective and scalable manner. However, targeting and delivery of LNPs across the blood-brain barrier (BBB) has proven challenging.

A SARS-CoV-2 targeted siRNA-nanoparticle therapy for COVID-19.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans. Despite several emerging vaccines, there remains no verifiable therapeutic targeted specifically to the virus. Here we present a highly effective siRNA therapeutic against SARS-CoV-2 infection using a novel lipid nanoparticle delivery system.

Mesenchymal Stem Cell exosome delivered Zinc Finger Protein activation of cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis is a genetic disorder that results in a multi-organ disease with progressive respiratory decline which leads to premature death. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene disrupts the capacity of the protein to function as a channel, transporting chloride ions and bicarbonate across epithelial cell membranes. Small molecule treatments targeted at potentiating or correcting CFTR have shown clinical benefits, but are only effective for a small percentage of individuals with specific CFTR mutations.

The Multifunctionality of Exosomes; from the Garbage Bin of the Cell to a Next Generation Gene and Cellular Therapy.

Exosomes are packaged with a variety of cellular cargo including RNA, DNA, lipids and proteins. For several decades now there has been ongoing debate as to what extent exosomes are the garbage bin of the cell or if these entities function as a distributer of cellular cargo which acts in a meaningful mechanistic way on target cells. Are the contents of exosomes unwanted excess cellular produce or are they selective nucleic acid packaged nanoparticles used to communicate in a paracrine fashion? Overexpressed RNAs and fragments of DNA have been shown to collect into exosomes which are jettisoned from cells in response to particular stimuli to maintain homeostasis suggesting exosomes are functional trash bins of the cell.

Designer nucleases to treat malignant cancers driven by viral oncogenes.

Viral oncogenic transformation of healthy cells into a malignant state is a well-established phenomenon but took decades from the discovery of tumor-associated viruses to their accepted and established roles in oncogenesis. Viruses cause ~ 15% of know cancers and represents a significant global health burden. Beyond simply causing cellular transformation into a malignant form, a number of these cancers are augmented by a subset of viral factors that significantly enhance the tumor phenotype and, in some cases, are locked in a state of oncogenic addiction, and substantial research has elucidated the mechanisms in these cancers providing a rationale for targeted inactivation of the viral components as a treatment strategy.