Nucleoside Modifications Suppress RNA Activation of Cytoplasmic RNA Sensors

Multiple innate defense pathways exist to recognize and defend against foreign nucleic acids. Unlike innate immune receptors that recognize structures specific for pathogens that are not shared by mammalian hosts — for example, toll-like receptor (TLR)4-lipopolysaccharide, TLR5-flagellin, NOD1 and 2-peptidoglycan — all nucleic acids are made from four components that are identical from bacteria to man. Nucleoside modifications are prevalent in nature but vary greatly in their distribution and frequency, and therefore could serve as patterns for recognition of pathogenic nucleic acids. The presence of modified nucleosides in RNA reduces the activation of RNA-sensing TLRs and retinoic acid inducible gene I (RIG-I), which initiate signaling cascades following activation and result in transcription of pro-inflammatory genes. Unexpectedly, translation of in vitro transcribed mRNA is enhanced by incorporation of modified nucleosides, but the mechanism responsible for this enhanced translation has not been identified. To identify the pathways responsible for enhanced translation of modified nucleoside-containing mRNA, we studied two cytoplasmic RNA-sensing innate defense mechanisms known to influence translation, the RNA-dependent protein kinase (PKR) pathway and the 2-5A system (oligoadenylate synthetase [OAS] and RNase L). Using purified protein in vitro, cell culture, and in vivo mouse studies, we show that unmodified in vitro transcribed mRNA activates PKR and OAS and is rapidly cleaved by RNase L. However, we show that incorporation of modified nucleosides into in vitro transcribed mRNA reduces each of these pathways. Furthermore, we demonstrate that these pathways are necessary for enhanced translation of mRNA containing modified nucleosides. Additionally, we demonstrate that the presence of pseudouridine in in vitro transcripts increases mRNA half-life following delivery. From these data, we conclude that unmodified in vitro transcribed mRNA is stimulatory to the cytoplasmic RNA sensors PKR and OAS. This stimulation is reduced by the presence of modified nucleosides. The enhanced translation of mRNA containing modified nucleosides results from reduced PKR and OAS activation. These data support a larger interpretation that the absence or reduction in frequency of modified nucleosides in RNA is a common pattern for recognition of pathogenic RNA by numerous innate defense systems

Autonomous Targeting of Infectious Superspreaders Using Engineered Transmissible Therapies

Infectious disease treatments, both pharmaceutical and vaccine, face three universal challenges: the difficulty of targeting treatments to high-risk ‘superspreader’ populations who drive the great majority of disease spread, behavioral barriers in the host population (such as poor compliance and risk disinhibition), and the evolution of pathogen resistance. Here, we describe a proposed intervention that would overcome these challenges by capitalizing upon Therapeutic Interfering Particles (TIPs) that are engineered to replicate conditionally in the presence of the pathogen and spread between individuals — analogous to ‘transmissible immunization’ that occurs with live-attenuated vaccines (but without the potential for reversion to virulence). Building on analyses of HIV field data from sub-Saharan Africa, we construct a multi-scale model, beginning at the single-cell level, to predict the effect of TIPs on individual patient viral loads and ultimately population-level disease prevalence. Our results show that a TIP, engineered with properties based on a recent HIV gene-therapy trial, could stably lower HIV/AIDS prevalence by ∼30-fold within 50 years and could complement current therapies. In contrast, optimistic antiretroviral therapy or vaccination campaigns alone could only lower HIV/AIDS prevalence by <2-fold over 50 years. The TIP's efficacy arises from its exploitation of the same risk factors as the pathogen, allowing it to autonomously penetrate superspreader populations, maintain efficacy despite behavioral disinhibition, and limit viral resistance. While demonstrated here for HIV, the TIP concept could apply broadly to many viral infectious diseases and would represent a new paradigm for disease control, away from pathogen eradication but toward robust disease suppression

HSV Recombinant Vectors for Gene Therapy

The very deep knowledge acquired on the genetics and molecular biology of herpes simplex virus (HSV), has allowed the development of potential replication-competent and replication-defective vectors for several applications in human healthcare. These include delivery and expression of human genes to cells of the nervous systems, selective destruction of cancer cells, prophylaxis against infection with HSV or other infectious diseases, and targeted infection to specific tissues or organs. Replication-defective recombinant vectors are non-toxic gene transfer tools that preserve most of the neurotropic features of wild type HSV-1, particularly the ability to express genes after having established latent infections, and are thus proficient candidates for therapeutic gene transfer settings in neurons. A replication-defective HSV vector for the treatment of pain has recently entered in phase 1 clinical trial. Replication-competent (oncolytic) vectors are becoming a suitable and powerful tool to eradicate brain tumours due to their ability to replicate and spread only within the tumour mass, and have reached phase II/III clinical trials in some cases. The progress in understanding the host immune response induced by the vector is also improving the use of HSV as a vaccine vector against both HSV infection and other pathogens. This review briefly summarizes the obstacle encountered in the delivery of HSV vectors and examines the various strategies developed or proposed to overcome such challenges

Computational Models of HIV-1 Resistance to Gene Therapy Elucidate Therapy Design Principles

Gene therapy is an emerging alternative to conventional anti-HIV-1 drugs, and can potentially control the virus while alleviating major limitations of current approaches. Yet, HIV-1's ability to rapidly acquire mutations and escape therapy presents a critical challenge to any novel treatment paradigm. Viral escape is thus a key consideration in the design of any gene-based technique. We develop a computational model of HIV's evolutionary dynamics in vivo in the presence of a genetic therapy to explore the impact of therapy parameters and strategies on the development of resistance. Our model is generic and captures the properties of a broad class of gene-based agents that inhibit early stages of the viral life cycle. We highlight the differences in viral resistance dynamics between gene and standard antiretroviral therapies, and identify key factors that impact long-term viral suppression. In particular, we underscore the importance of mutationally-induced viral fitness losses in cells that are not genetically modified, as these can severely constrain the replication of resistant virus. We also propose and investigate a novel treatment strategy that leverages upon gene therapy's unique capacity to deliver different genes to distinct cell populations, and we find that such a strategy can dramatically improve efficacy when used judiciously within a certain parametric regime. Finally, we revisit a previously-suggested idea of improving clinical outcomes by boosting the proliferation of the genetically-modified cells, but we find that such an approach has mixed effects on resistance dynamics. Our results provide insights into the short- and long-term effects of gene therapy and the role of its key properties in the evolution of resistance, which can serve as guidelines for the choice and optimization of effective therapeutic agents