Advances in HIV Gene Therapy.

Early gene therapy studies held great promise for the cure of heritable diseases, but the occurrence of various genotoxic events led to a pause in clinical trials and a more guarded approach to progress. Recent advances in genetic engineering technologies have reignited interest, leading to the approval of the first gene therapy product targeting genetic mutations in 2017. Gene therapy (GT) can be delivered either in vivo or ex vivo. An ex vivo approach to gene therapy is advantageous, as it allows for the characterization of the gene-modified cells and the selection of desired properties before patient administration. Autologous cells can also be used during this process which eliminates the possibility of immune rejection. This review highlights the various stages of ex vivo gene therapy, current research developments that have increased the efficiency and safety of this process, and a comprehensive summary of Human Immunodeficiency Virus (HIV) gene therapy studies, the majority of which have employed the ex vivo approach.

Layer-by-Layer Particles Deliver Epigenetic Silencing siRNA to HIV-1 Latent Reservoir Cell Types.

For over two decades, nanomaterials have been employed to facilitate intracellular delivery of small interfering RNA (siRNA), both in vitro and in vivo, to induce post-transcriptional gene silencing (PTGS) via RNA interference. Besides PTGS, siRNAs are also capable of transcriptional gene silencing (TGS) or epigenetic silencing, which targets the gene promoter in the nucleus and prevents transcription via repressive epigenetic modifications. However, silencing efficiency is hampered by poor intracellular and nuclear delivery. Here, polyarginine-terminated multilayered particles are reported as a versatile system for the delivery of TGS-inducing siRNA to potently suppress virus transcription in HIV-infected cells. siRNA is complexed with multilayered particles formed by layer-by-layer assembly of poly(styrenesulfonate) and poly(arginine) and incubated with HIV-infected cell types, including primary cells. Using deconvolution microscopy, uptake of fluorescently labeled siRNA is observed in the nuclei of HIV-1 infected cells. Viral RNA and protein are measured to confirm functional virus silencing from siRNA delivered using particles 16 days post-treatment. This work extends conventional particle-enabled PTGS siRNA delivery to the TGS pathway and paves the way for future studies on particle-delivered siRNA for efficient TGS of various diseases and infections, including HIV.

Nanoparticle Delivery Platforms for RNAi Therapeutics Targeting COVID-19 Disease in the Respiratory Tract.

Since December 2019, a pandemic of COVID-19 disease, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly spread across the globe. At present, the Food and Drug Administration (FDA) has issued emergency approval for the use of some antiviral drugs. However, these drugs still have limitations in the specific treatment of COVID-19, and as such, new treatment strategies urgently need to be developed. RNA-interference-based gene therapy provides a tractable target for antiviral treatment. Ensuring cell-specific targeted delivery is important to the success of gene therapy. The use of nanoparticles (NPs) as carriers for the delivery of small interfering RNA (siRNAs) to specific tissues or organs of the human body could play a crucial role in the specific therapy of severe respiratory infections, such as COVID-19. In this review, we describe a variety of novel nanocarriers, such as lipid NPs, star polymer NPs, and glycogen NPs, and summarize the pre-clinical/clinical progress of these nanoparticle platforms in siRNA delivery. We also discuss the application of various NP-capsulated siRNA as therapeutics for SARS-CoV-2 infection, the challenges with targeting these therapeutics to local delivery in the lung, and various inhalation devices used for therapeutic administration. We also discuss currently available animal models that are used for preclinical assessment of RNA-interference-based gene therapy. Advances in this field have the potential for antiviral treatments of COVID-19 disease and could be adapted to treat a range of respiratory diseases.

RNA-induced epigenetic silencing inhibits HIV-1 reactivation from latency.

BACKGROUND: Current antiretroviral therapy is effective in controlling HIV-1 infection. However, cessation of therapy is associated with rapid return of viremia from the viral reservoir. Eradicating the HIV-1 reservoir has proven difficult with the limited success of latency reactivation strategies and reflects the complexity of HIV-1 latency. Consequently, there is a growing need for alternate strategies. Here we explore a "block and lock" approach for enforcing latency to render the provirus unable to restart transcription despite exposure to reactivation stimuli. Reactivation of transcription from latent HIV-1 proviruses can be epigenetically blocked using promoter-targeted shRNAs to prevent productive infection. We aimed to determine if independent and combined expression of shRNAs, PromA and 143, induce a repressive epigenetic profile that is sufficiently stable to protect latently infected cells from HIV-1 reactivation when treated with a range of latency reversing agents (LRAs). RESULTS: J-Lat 9.2 cells, a model of HIV-1 latency, expressing shRNAs PromA, 143, PromA/143 or controls were treated with LRAs to evaluate protection from HIV-1 reactivation as determined by levels of GFP expression. Cells expressing shRNA PromA, 143, or both, showed robust resistance to viral reactivation by: TNF, SAHA, SAHA/TNF, Bryostatin/TNF, DZNep, and Chaetocin. Given the physiological importance of TNF, HIV-1 reactivation was induced by TNF (5 ng/mL) and ChIP assays were performed to detect changes in expression of epigenetic markers within chromatin in both sorted GFP- and GFP+ cell populations, harboring latent or reactivated proviruses, respectively. Ordinary two-way ANOVA analysis used to identify interactions between shRNAs and chromatin marks associated with repressive or active chromatin in the integrated provirus revealed significant changes in the levels of H3K27me3, AGO1 and HDAC1 in the LTR, which correlated with the extent of reduced proviral reactivation. The cell line co-expressing shPromA and sh143 consistently showed the least reactivation and greatest enrichment of chromatin compaction indicators. CONCLUSION: The active maintenance of epigenetic silencing by shRNAs acting on the HIV-1 LTR impedes HIV-1 reactivation from latency. Our "block and lock" approach constitutes a novel way of enforcing HIV-1 "super latency" through a closed chromatin architecture that renders the virus resistant to a range of latency reversing agents.

Direct evidence of nuclear Argonaute distribution during transcriptional silencing links the actin cytoskeleton to nuclear RNAi machinery in human cells.

Mammalian RNAi machinery facilitating transcriptional gene silencing (TGS) is the RNA-induced transcriptional gene silencing-like (RITS-like) complex, comprising of Argonaute (Ago) and small interfering RNA (siRNA) components. We have previously demonstrated promoter-targeted siRNA induce TGS in human immunodeficiency virus type-1 (HIV-1) and simian immunodeficiency virus (SIV), which profoundly suppresses retrovirus replication via heterochromatin formation and histone methylation. Here, we examine subcellular co-localization of Ago proteins with promoter-targeted siRNAs during TGS of SIV and HIV-1 infection. Analysis of retrovirus-infected cells revealed Ago1 co-localized with siRNA in the nucleus, while Ago2 co-localized with siRNA in the inner nuclear envelope. Mismatched and scrambled siRNAs were observed in the cytoplasm, indicating sequence specificity. This is the first report directly visualizing nuclear compartment distribution of Ago-associated siRNA and further reveals a novel nuclear trafficking mechanism for RITS-like components involving the actin cytoskeleton. These results establish a model for elucidating mammalian TGS and suggest a fundamental mechanism underlying nuclear delivery of RITS-like components.

Current State of Therapeutics for HTLV-1.

Human T cell leukaemia virus type-1 (HTLV-1) is an oncogenic retrovirus that causes lifelong infection in ~5-10 million individuals globally. It is endemic to certain First Nations populations of Northern and Central Australia, Japan, South and Central America, Africa, and the Caribbean region. HTLV-1 preferentially infects CD4+ T cells and remains in a state of reduced transcription, often being asymptomatic in the beginning of infection, with symptoms developing later in life. HTLV-1 infection is implicated in the development of adult T cell leukaemia/lymphoma (ATL) and HTLV-1-associated myelopathies (HAM), amongst other immune-related disorders. With no preventive or curative interventions, infected individuals have limited treatment options, most of which manage symptoms. The clinical burden and lack of treatment options directs the need for alternative treatment strategies for HTLV-1 infection. Recent advances have been made in the development of RNA-based antiviral therapeutics for Human Immunodeficiency Virus Type-1 (HIV-1), an analogous retrovirus that shares modes of transmission with HTLV-1. This review highlights past and ongoing efforts in the development of HTLV-1 therapeutics and vaccines, with a focus on the potential for gene therapy as a new treatment modality in light of its successes in HIV-1, as well as animal models that may help the advancement of novel antiviral and anticancer interventions.

The interaction between Epstein-Barr virus and multiple sclerosis genetic risk loci: insights into disease pathogenesis and therapeutic opportunities.

Multiple sclerosis (MS) is a chronic neurodegenerative autoimmune disease, characterised by the demyelination of neurons in the central nervous system. Whilst it is unclear what precisely leads to MS, it is believed that genetic predisposition combined with environmental factors plays a pivotal role. It is estimated that close to half the disease risk is determined by genetic factors. However, the risk of developing MS cannot be attributed to genetic factors alone, and environmental factors are likely to play a significant role by themselves or in concert with host genetics. Epstein-Barr virus (EBV) infection is the strongest known environmental risk factor for MS. There has been increasing evidence that leaves little doubt that EBV is necessary, but not sufficient, for developing MS. One plausible explanation is EBV may alter the host immune response in the presence of MS risk alleles and this contributes to the pathogenesis of MS. In this review, we discuss recent findings regarding how EBV infection may contribute to MS pathogenesis via interactions with genetic risk loci and discuss possible therapeutic interventions.

Human papillomavirus in the setting of immunodeficiency: Pathogenesis and the emergence of next-generation therapies to reduce the high associated cancer risk.

Human papillomavirus (HPV), a common sexually transmitted virus infecting mucosal or cutaneous stratified epithelia, is implicated in the rising of associated cancers worldwide. While HPV infection can be cleared by an adequate immune response, immunocompromised individuals can develop persistent, treatment-refractory, and progressive disease. Primary immunodeficiencies (PIDs) associated with HPV-related disease include inborn errors of GATA, EVER1/2, and CXCR4 mutations, resulting in defective cellular function. People living with secondary immunodeficiency (e.g. solid-organ transplants recipients of immunosuppression) and acquired immunodeficiency (e.g. concurrent human immunodeficiency virus (HIV) infection) are also at significant risk of HPV-related disease. Immunocompromised people are highly susceptible to the development of cutaneous and mucosal warts, and cervical, anogenital and oropharyngeal carcinomas. The specific mechanisms underlying high-risk HPV-driven cancer development in immunocompromised hosts are not well understood. Current treatments for HPV-related cancers include surgery with adjuvant chemotherapy and/or radiotherapy, with clinical trials underway to investigate the use of anti-PD-1 therapy. In the setting of HIV co-infection, persistent high-grade anal intraepithelial neoplasia can occur despite suppressive antiretroviral therapy, resulting in an ongoing risk for transformation to overt malignancy. Although therapeutic vaccines against HPV are under development, the efficacy of these in the setting of PID, secondary- or acquired- immunodeficiencies remains unclear. RNA-based therapeutic targeting of the HPV genome or mRNA transcript has become a promising next-generation therapeutic avenue. In this review, we summarise the current understanding of HPV pathogenesis, immune evasion, and malignant transformation, with a focus on key PIDs, secondary immunodeficiencies, and HIV infection. Current management and vaccine regimes are outlined in relation to HPV-driven cancer, and specifically, the need for more effective therapeutic strategies for immunocompromised hosts. The recent advances in RNA-based gene targeting including CRISPR and short interfering RNA (siRNA), and the potential application to HPV infection are of great interest. An increased understanding of both the dysregulated immune responses in immunocompromised hosts and of viral persistence is essential for the design of next-generation therapies to eliminate HPV persistence and cancer development in the most at-risk populations.

Novel siRNA therapeutics demonstrate multi-variant efficacy against SARS-CoV-2.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a respiratory virus that causes COVID-19 disease, with an estimated global mortality of approximately 2%. While global response strategies, which are predominantly reliant on regular vaccinations, have shifted from zero COVID to living with COVID, there is a distinct lack of broad-spectrum direct acting antiviral therapies that maintain efficacy across evolving SARS-CoV-2 variants of concern. This is of most concern for immunocompromised and immunosuppressed individuals who lack robust immune responses following vaccination, and others at risk for severe COVID and long-COVID. RNA interference (RNAi) therapeutics induced by short interfering RNAs (siRNAs) offer a promising antiviral treatment option, with broad-spectrum antiviral capabilities unparalleled by current antiviral therapeutics and a high genetic barrier to antiviral escape. Here we describe novel siRNAs, targeting highly conserved regions of the SARS-CoV-1 and 2 genome of both human and animal species, with multi-variant antiviral potency against eight SARS-CoV-2 lineages - Ancestral VIC01, Alpha, Beta, Gamma, Delta, Zeta, Kappa and Omicron. Treatment with our siRNA resulted in significant protection against virus-mediated cell death in vitro, with >97% cell survival (P < 0.0001), and corresponding reductions of viral nucleocapsid RNA of up to 99.9% (P < 0.0001). When compared to antivirals; Sotrovimab and Remdesivir, the siRNAs demonstrated a more potent antiviral effect and similarly, when multiplexing siRNAs to target different viral regions simultaneously, an increased antiviral effect was observed compared to individual siRNA treatments (P < 0.0001). These results demonstrate the potential for a highly effective broad-spectrum direct acting antiviral against multiple SARS-CoV-2 variants, including variants resistant to antivirals and vaccine generated neutralizing antibodies.

Targeted Nanocarrier Delivery of RNA Therapeutics to Control HIV Infection.

Our understanding of HIV infection has greatly advanced since the discovery of the virus in 1983. Treatment options have improved the quality of life of people living with HIV/AIDS, turning it from a fatal disease into a chronic, manageable infection. Despite all this progress, a cure remains elusive. A major barrier to attaining an HIV cure is the presence of the latent viral reservoir, which is established early in infection and persists for the lifetime of the host, even during prolonged anti-viral therapy. Different cure strategies are currently being explored to eliminate or suppress this reservoir. Several studies have shown that a functional cure may be achieved by preventing infection and also inhibiting reactivation of the virus from the latent reservoir. Here, we briefly describe the main HIV cure strategies, focussing on the use of RNA therapeutics, including small interfering RNA (siRNA) to maintain HIV permanently in a state of super latency, and CRISPR gRNA to excise the latent reservoir. A challenge with progressing RNA therapeutics to the clinic is achieving effective delivery into the host cell. This review covers recent nanotechnological strategies for siRNA delivery using liposomes, N-acetylgalactosamine conjugation, inorganic nanoparticles and polymer-based nanocapsules. We further discuss the opportunities and challenges of those strategies for HIV treatment.

Nanoscale probing and imaging of HIV-1 RNA in cells with a chimeric LNA-DNA sensor.

Real-time detection and nanoscale imaging of human immunodeficiency virus type 1 ribonucleic acid (HIV-1 RNA) in latently infected cells that persist in people living with HIV-1 on antiretroviral therapy in blood and tissue may reveal new insights needed to cure HIV-1 infection. Herein, we develop a strategy combining DNA nanotechnology and super-resolution expansion microscopy (ExM) to detect and image a 22 base sequence transcribed from the HIV-1 promoter in model live and fixed cells. We engineer a chimeric locked nucleic acid (LNA)-DNA sensor via hybridization chain reaction to probe HIV-1 RNA in the U3 region of the HIV-1 long terminal repeat (LTR) by signal amplification in live cells. We find that the viral RNA transcript of the U3 region of the HIV-1 LTR, namely PromA, is a valid and specific biomarker to detect infected live cells. The efficiency and selectivity of the LNA-DNA sensor are evaluated in combination with ExM. Unlike standard ExM methods, which rely on additional custom linkers to anchor and immobilize RNA molecules in the intracellular polymeric network, in the current strategy, we probe and image the HIV-1 RNA target at nanoscale resolution, without resorting to chemical linkers or additional preparation steps. This is achieved by physical entrapment of the HIV-1 viral transcripts in the cells post-expansion by finely tuning the mesh size of the intracellular polymeric network.

Transcriptional gene silencing of HIV-1 through promoter targeted RNA is highly specific.

We have previously reported induction of transcriptional gene silencing (TGS) of HIV-1 by short hairpin RNA (shRNA) expressed in MOLT-4 cells. The shRNA (termed shPromA) targets the highly conserved tandem NF-kB binding sequences of the HIV-1 promoter. Recent articles have reported that TGS mediated by promoter-targeted siRNAs was exclusively the result of sequence non-specific off-target effects. Specifically, several mismatched siRNAs to the target promoter sequences were reported to also induce significant TGS, suggesting TGS was a consequence of off-target effects. Here, following extensive investigation, we report that shPromA induces sequence specific transcriptional silencing in HIV-1 infection in MOLT4 cells, while four shRNA variants, mismatched by 2-3 nucleotides, fail to suppress viral replication. We confirm similar levels of shRNA expression from the U6 promoter and the presence of processed/cleaved siRNAs for each construct in transduced MOLT-4 cells. HIV-1 sequence specific shPromA does not suppress HIV-2, which has an alternate NF-kB binding sequence. As a result of the unique sequence targeted, shPromA does not induce down-regulation of other NF-kB driven genes, either at the mRNA or protein level. Furthermore, we confirmed shPromA does not have sequence non-specific off-target effects through unaltered expression of CD4, CXCR4, and CCR5, which are used for viral entry. Additionally, shPromA does not alter PKR, IFN levels, and three downstream mediators of IFN-a response genes. Our data clearly shows that shPromA achieved highly specific TGS of HIV-1, demonstrating that effective TGS can be induced with minimal off-target effects.

Block and Lock HIV Cure Strategies to Control the Latent Reservoir.

The HIV latent reservoir represents the major challenge to cure development. Residing in resting CD4+ T cells and myeloid cells at multiple locations in the body, including sanctuary sites such as the brain, the latent reservoir is not eliminated by ART and has the ability to reactivate virus replication to pre-therapy levels when ART is ceased. There are four broad areas of HIV cure research. The only successful cure strategy, thus far, is stem cell transplantation using naturally HIV resistant CCR5Δ32 stem cells. A second potential cure approach uses gene editing technology, such as zinc-finger nucleases and CRISPR/Cas9. Another two cure strategies aim to control the HIV reservoir, with polar opposite concepts; The "shock and kill" approach, which aims to "shock" or reactivate the latent virus and then "kill" infected cells via targeted immune responses. Lastly, the "block and lock" approach, which aims to enhance the latent virus state by "blocking" HIV transcription and "locking" the HIV promoter in a deep latent state via epigenetic modifications. "Shock and kill" approaches are a major focus of cure studies, however we predict that the increased specificity of "block and lock" approaches will be required for the successful development of a sustained HIV clinical remission in the absence of ART. This review focuses on the current research of novel "block and lock" approaches being explored to generate an HIV cure via induction of epigenetic silencing. We will also discuss potential future therapeutic delivery and the challenges associated with progressing "block and lock" cure approaches as these move toward clinical trials.

RNAi therapeutics: an antiviral strategy for human infections.

Gene silencing induced by RNAi represents a promising antiviral development strategy. This review will summarise the current state of RNAi therapeutics for treating acute and chronic human virus infections. The gene silencing pathways exploited by RNAi therapeutics will be described and include both classic RNAi, inducing cytoplasmic mRNA degradation post-transcription and novel RNAi, mediating epigenetic modifications at the transcription level in the nucleus. Finally, the challenge of delivering gene modifications via RNAi will be discussed, along with the unique characteristics of respiratory versus systemic administration routes to highlight recent advances and future potential of RNAi antiviral treatment strategies.

Delivery of gene therapy to resting immune cells for an HIV cure.

PURPOSE OF REVIEW: The utilization of genetically modified T cells to therapeutically target to various previously incurable diseases such, as cancer, has expanded exponentially in recent years. This success now provides the motivating force in applying the same technology for incurable infectious diseases including HIV. The common bottleneck in gene therapy continues to be at the level of gene delivery. Although present approaches adapt the cell to the delivery technology, emerging techniques now focus on leaving cells in their phenotypically resting state. In doing so, engraftment and proliferation potential are retained and in turn increase the efficacy of this approach at a lowered cost. This review will outline the main efforts of gene delivery using viral vectors or nonviral vectors and challenges moving forward not only in resting T cells, but also in other resting immune cells including hematopoietic stem cells. RECENT FINDINGS: In focusing on HIV cure efforts using gene therapy, progress on solving the challenges of gene delivery will be described for both viral and nonviral vectors. Advances in the basic virology of lentiviruses have led to the proposal of many next generation lentiviral vector platforms for resting immune cells. Moreover, we will also highlight the progress made in nonviral approaches using nanotechnology as alternatives and/or synergistic technologies to be used alongside lentiviral platforms. SUMMARY: The innovative approaches described in these recent studies, particularly those using the natural mechanisms employed by HIV to enhance for example virus entry or virus latency, will enable future optimization of gene delivery platforms and therapeutics, which will importantly, provide a pathway toward translation into clinical practice.

The Role of Zinc in Antiviral Immunity.

Zinc is an essential trace element that is crucial for growth, development, and the maintenance of immune function. Its influence reaches all organs and cell types, representing an integral component of approximately 10% of the human proteome, and encompassing hundreds of key enzymes and transcription factors. Zinc deficiency is strikingly common, affecting up to a quarter of the population in developing countries, but also affecting distinct populations in the developed world as a result of lifestyle, age, and disease-mediated factors. Consequently, zinc status is a critical factor that can influence antiviral immunity, particularly as zinc-deficient populations are often most at risk of acquiring viral infections such as HIV or hepatitis C virus. This review summarizes current basic science and clinical evidence examining zinc as a direct antiviral, as well as a stimulant of antiviral immunity. An abundance of evidence has accumulated over the past 50 y to demonstrate the antiviral activity of zinc against a variety of viruses, and via numerous mechanisms. The therapeutic use of zinc for viral infections such as herpes simplex virus and the common cold has stemmed from these findings; however, there remains much to be learned regarding the antiviral mechanisms and clinical benefit of zinc supplementation as a preventative and therapeutic treatment for viral infections.

Zinc is a potent and specific inhibitor of IFN-λ3 signalling.

Lambda interferons (IFNL, IFN-λ) are pro-inflammatory cytokines important in acute and chronic viral infection. Single-nucleotide polymorphisms rs12979860 and rs8099917 within the IFNL gene locus predict hepatitis C virus (HCV) clearance, as well as inflammation and fibrosis progression in viral and non-viral liver disease. The underlying mechanism, however, is not defined. Here we show that the rs12979860 CC genotype correlates with increased hepatic metallothionein expression through increased systemic zinc levels. Zinc interferes with IFN-λ3 binding to IFNL receptor 1 (IFNLR1), resulting in decreased antiviral activity and increased viral replication (HCV, influenza) in vitro. HCV patients with high zinc levels have low hepatocyte antiviral and inflammatory gene expression and high viral loads, confirming the inhibitory role of zinc in vivo. We provide the first evidence that zinc can act as a potent and specific inhibitor of IFN-λ3 signalling and highlight its potential as a target of therapeutic intervention for IFN-λ3-mediated chronic disease.