Using iPSC variation to define HIV-1 regulatory networks

The pathogenic retrovirus human immunodeficiency virus type 1 (HIV-1) has caused the worldwide acquired immunodeficiency syndrome (AIDS) pandemic. It is estimated that ~37 million people currently live with HIV-1, with ~1.8 million new cases of infection and ~1 million AIDS deaths occurring every year (2017 figures, source: http://www.unaids.org/en/ resources/fact-sheet). AIDS is currently considered a treatable disease as a battery of effective anti-viral drugs that target essential viral proteins have been developed. Critically, there is no vaccine to prevent new infections and, once a person is infected, there is no cure (treatment leading to viral eradication). Moreover, with the passage of time, drug-resistant strains are becoming an increasingly significant problem in the clinic. It is therefore necessary to develop new anti-HIV-1 therapeutic approaches.

Despite many years of intense investigation, the understanding of the complexity of cell-encoded factors and pathways that regulate HIV-1 replication (both positively and negatively) remains significantly incomplete. The identification and definition of new viral regulatory factors will suggest novel targets for future therapeutic intervention which, in addition to helping control viral growth and HIV-1-associated disease, may also impact research programmes seeking HIV-1 cure and/or vaccination. The feasibility of therapeutically targeting a cellular protein to inhibit HIV-1 is demonstrated by Maraviroc, a small molecule inhibitor that binds the HIV-1 entry receptor CCR5 to block infection.

In this programme, we will develop and exploit an innovative inter-disciplinary strategy to discover novel human cell factors and pathways that regulate HIV-1 replication. We will use the deeply characterised HipSci library (www.hipsci.org) of induced pluripotent stem cells (iPSCs), for which detailed genomic, transcriptomic and proteomic information already exists, and correlate variations in HIV-1 infection phenotypes with key molecular signatures that are intrinsic to each iPSC line. To complement this approach, we will also reprogramme blood samples from patients at high risk of HIV-1 infection, and with known disease outcomes (the Multicenter AIDS Cohort Study - MACS; http://aidscohortstudy.org/), into iPSCs. Co-ordinated characterisation of these lines will add a further dimension to our study by also enabling molecular signatures and infection data to be correlated with clinical outcomes. In depth bioinformatic interrogation of these orthogonal datasets will suggest candidate cell-encoded HIV-1 regulatory factors, which will then be tested for function using established laboratory models of HIV-1 infection.

In sum, our experimental pipeline comprises: (i) screening iPSC cell lines from two sources to find extreme HIV-1 infection phenotypes, (ii) using bioinformatic tools and whole cell molecular signatures to discover new regulatory factors and (iii) validating our findings in cell culture systems and characterising the mechanisms of action of novel HIV-1 regulators at the molecular level. Our goal is to uncover novel human proteins and pathways controlling HIV-1 replication, and to inform initiatives aiming to devise fresh therapeutic strategies for overcoming HIV-1 infection. Finally, and importantly, we are committed to sharing the iPSC lines that we will derive from HIV-1 exposed individuals: these can serve as a unique resource to study HIV-1 biology since iPSCs are virtually inexhaustible and have the potential to be differentiated into any human cell type, including the natural cellular targets of HIV-1.

The goal of our programme is to discover, validate and characterise new HIV-1 regulatory factors and pathways. To this end, we will:

1) Screen human induced pluripotent stem cells (iPSCs) from the HipSci library and iPSCs that we will generate from clinical samples (from the Multicentre AIDS Cohort Study, MACS) for HIV-1 infection and replication phenotypes. HIV-1 infectivity will be assessed using a lentiviral vector expressing GFP and detected by microscopy and flow cytometry. Replication will be assessed by viral p24Gag release and progeny infectivity. We will use established assays to map the affected step(s) in the HIV-1 life cycle in cell lines presenting extreme replication phenotypes.

2) Correlate extreme phenotypes with molecular signatures of the cell lines at the genomic, transcriptomic and proteomic level, using bioinformatic tools to identify candidate regulatory host factors and pathways. We will leverage from experience in multi-dimensional reduction of phenotypic datasets using Bayesian algorithms tools such as Probabilistic Estimation of Expression Residuals (PEER) to integrate cell behaviour with -omics datasets.

3) Validate HIV-1-regulatory factors and pathways in cultured cells, CD4+ T cells and macrophages, by down regulation (shRNA, siRNA and CRIPSR/cas9) or ectopic overexpression of candidate genes. We will characterise the mechanism(s) of action using technologies that align, initially, with predicted biological activities. We will also repurpose existing drugs that target the newly discovered factors/pathways to test their effect on HIV-1 replication. Lastly, we will investigate the association of regulatory factors with AIDS clinical outcomes and disease progression from existing and emerging patient cohort data, such as the MACS.

This work seeks to inform new therapeutic strategies to treat HIV-1.

This research programme aims to identify novel human factors that regulate HIV-1 replication, and to define their mechanisms of action. Deeper insights into the molecular interactions between HIV-1 and its human host can offer therapeutic opportunities to suppress virus spread and disease. While this is a discovery-focused investigation to identify novel cell-encoded factors, the scientific as well as the economic, health and social impact of the findings can be broad.

People infected with HIV-1 require a lifetime course of highly-active antiretroviral treatment (HAART) to control the virus clinically. Despite being effective, the mixture of antiretroviral drugs, while inhibiting viral replication and supporting normal life expectancy, presents a substantial cost to the NHS (lifetime HAART costs ~£0.3m). The burden for the patient is also non-negligible, as they require daily medication. Though HAART limits viral replication effectively, it does not eradicate HIV-1, in large part because the viral genome is converted to DNA that stably integrates into host chromosomal DNA establishing a state of lifelong persistence. Accordingly, there is great interest in seeking to achieve HIV-1 eradication (cure), and this is presumed to require both purposeful activation/emergence of the virus from its persistent state, and the prevention of new rounds of infection ("kick-and-kill"). This is an extremely challenging ambition, and the development of new pharmacologic agents that can participate in viral activation or neutralisation will add fresh perspectives and tools to this important problem. To develop novel therapeutics rationally, it will be valuable to consider all viral regulatory mechanisms and pathways, and this will be facilitated by possessing a more comprehensive understanding of the scope of host-HIV-1 interactions (stimulatory as well as repressive) that regulate the viral life cycle.

Here we outline a novel inter-disciplinary strategy that exploits natural variation in the behaviour of cells derived from different people to identify and understand cellular factors and pathways that modulate HIV-1 replication. By finding new host proteins and mechanisms that regulate HIV-1, we will not only increase the fundamental knowledge base regarding this deadly human pathogen, but we will provide scientists in academic and commercial organisations with molecules for consideration as potential future drug targets. Moreover, we will explore the repurposing of available drugs that target the factors we identify as lead anti-viral inhibitors. Our screening pipeline can also be readily applied to other fields: for instance, the regulation of other viruses or noxious stimuli by the host can be interrogated once a reproducible mid-level throughput phenotyping assay has been established.

This programme will also generate a new library of induced pluripotent stem cells from patients who have been multiply exposed to HIV-1 and whose clinical outcomes have been recorded in great detail. These cells will be made available to the scientific community and will provide an effectively inexhaustible and invaluable resource to study HIV-1 biology, where cultured cell infection phenotypes can be mapped onto patient natural histories.

Finally, greater understanding of HIV-1 infection of iPS cells and the hurdles that the virus has to overcome to infect these cells can inform many areas of research that utilise HIV-1-based (lenti-)vectors to deliver genetic material to cells. Our work has the potential to lead to improvements in vector design and transduction protocols, and may therefore benefit translationally-focused areas of science such as gene therapy and regenerative medicine.