Targeting senescence-specific apoptosis resistance genes for the clearance of senescent cells in 2D and 3D human models

Senescence is the process of biological ageing that takes place in living cells and the abundance of senescent cells increase in our bodies as we age. As many people will be aware, age is one of the biggest risk factors for a number of diseases, and it is thought that senescent cells and the factors that they secrete may drive the ageing process. Controlled and targeted clearance of the senescent cell burden may have considerable health benefits and is an attractive strategy for extending our healthspan. This research programme seeks to identify new gene that can be targeted for clearance of senescent cells and identify novel compounds to achieve this aim.

Until very recently it was not possible to perform targeted gene knockdown in senescent cells. However, the applicant has successfully developed a technique to turn individual genes off in senescent cells. This has enabled us to screen for genes that senescent cells have become 'addicted to' and find those that can be targeted to specifically kill these aged cells. Using this strategy, we have identified 16 such genes. Excitingly, these genes appear to be functionally related, interact with key drivers of senescence and are observed to increase their expression in a range of cells and tissues with biological ageing.

Our bodies are made up of lots of different cell types, and an important first step will be to determine if turning these genes off in different cell types also results in the specific clearance of senescent cells. This will involve the culture of epithelial cells and fibroblasts from two different tissues, breast and skin. These cells have already been grown and aged in our laboratory. We will also validate the reported increase in expression with age using skin biopsies. Throughout this work, we will examine a range of known senescence markers, determine the precise cell death pathways that are engaged and check that our approaches are well tolerated by 'healthy' young cells. We will use a mixture of sophisticated, automated microscopy techniques coupled with molecular biology and others assays to achieve these aims.

It is anticipated that future strategies for the targeted removal of senescent cells (so call senolytic therapies) will involve intermittent administration during periods of good health. We will test a range of different regimes to model the optimal treatment strategy for human senescent cell clearance. In addition to turning off our selected genes, we will perform a small-scale compound screen to see if we can identify senolytic compounds which inhibit our genes of interest. Next, we will use state of the art 3D co-culture systems to generate 'skin in a dish' and determine the impact of senescent cell clearance on skin ageing phenotypes. Finally, we will perform a high-throughput compound screen to identify novel compounds that can achieve targeted senescent cell clearance in cells in a dish and in our 3D models. Results of this research programme will therefore provide much needed information about the route to senescent cell clearance, identify novel senolytic compounds and bespoke treatment regimes. We hope our research will pave the way for the development of future treatments for senescent cell clearance.

This work will test the following hypotheses
1. SSAR genes can be targeted to induce senescent cell death (clearance) in multiple cell types and tissues.
2. Regular clearance of senescent cells, either by siRNA/shRNA/small molecules, will remove inherent paracrine senescence present in 2D cultures leading to extended culture lifespan.
3. Clearance of senescent cells in 3D skin organotypic co-culture systems (OCSs) will alter skin ageing phenotypes.
4. Small molecule inhibition of SSAR genes can suppress the effects of senescence either by SASP protection or by targeted cell death.

We will validate the route to senescent-specific apoptosis in a panel of cell types using qRTPCR, western blot analysis/immunofluorescence, apoptosis assays and readouts for SASP protection. In vivo validation will be performed using immunohistochemistry for SSAR proteins and senescence markers in human skin samples from a range of aged individuals.

Inducible shRNA models will be generated. New routes for the extension of lifespan will be sought by altering 'treatment regimes' with shRNAs and putative senolytic compounds which inhibit SSARs. 3D skin OCSs will be used to model senescent cell clearance in 3D and the consequences of shRNA knockdown/senolytic application. Readouts for skin ageing phenotypes (thickness, folding, barrier function, wound healing and pro-collagen 1) will be employed.

Finally, we will perform a large-scale compound screen in 2D with 3D validation for novel senolytic compounds. This work will involve high-throughput liquid handing and high content automated microscopy of EP and DS cell throughout. Images will be quantitated using sophisticated image analysis protocols (IN Cell Developer analysis software, V1.9, GE). Data will be generated in the form of .lg3 and .xls files (see Data Management plan).

This project aims to uncover new routes to reduce the senescent cell burden via gene modulation and to identify new small molecule 'senolytics' for the targeted killing of senescent cells. Mounting evidence implicates senescence in a number of ageing-associated diseases such as type 2 diabetes, cancer and atherosclerosis. This work will make a significant contribution to our understanding of the fundamental process of senescence. At the same time, through providing new knowledge to advance the development of interventions to improve human health and wellbeing, this work can contribute to the wider goal of promoting healthy ageing. As such, this proposal aligns to the BBSRC's Key Research Priority 3: Bioscience for health (Strategic plan 2013-2014), under the umbrella of 'the ageing process' and 'new regenerative biology and tissue engineering' research opportunities.

Academia
Besides the impact on the scientific community (see Academic Beneficiaries), the staff employed during this grant will be immediate beneficiaries (see Pathways to Impact). Working closely with Unilever, this will be achieved through their continued professional development, knowledge exchange, and the preparation of Fellowship applications and manuscripts for publication.

Business and Industry
We anticipate that outcomes from this project will have impact on the Economy and the Society. This work will contribute to UK economic competitiveness through the training of highly-skilled people with the needs of biopharma industry in mind thanks to the collaboration with Unilever. The senolytic molecules that emerge from this work will be amenable to potential commercial exploitation by our industrial partner, Unilever. As detailed in the Case for support, Unilever's products are used by 2 billion consumers every day and they have the necessary infrastructure to translate the findings of this proposal into fast-moving consumer goods.

The 2D and 3D modules that will be developed and refined as part of this work will be of broad interest for compound screening applications (both within academia and industry). As an example, there is growing evidence that senescent cells are generated and retained as a consequence of radiotherapy treatment. Future cancer therapies may require combinatorial strategies that enable the elimination of these 'byproducts' of current treatment regimes. The development of advanced 3D cell models for compound testing also has the potential to reduce the number of animals used in future research endeavours and improve the quality of research results.

The General Public
We will continue to actively engage with non-academic beneficiaries through public engagement, via the Media and Public Relations department at QMUL/Unilever. Lay information will be disseminated through product marketing information as well as trade articles to benefit the general public, dermatologists and other medical professions (see Case for Support, Benefits to the wider economy and society, page 8).