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BGRF and SILS Scientists Analyze Viability of shRNA Therapy for Huntington’s Disease – Press Release by Biogerontology Research Foundation

BGRF and SILS Scientists Analyze Viability of shRNA Therapy for Huntington’s Disease – Press Release by Biogerontology Research Foundation

Biogerontology Research Foundation


Friday, December 1, 2017, London, UK: Researchers from the Biogerontology Research FoundationDepartment of Molecular Neuroscience at the Swammerdam Institute for Life Sciences at the University of Amsterdam, and the Department of Neurobiology, Care Sciences and Society at the Karolinska Institute announce the publication of a paper in Translational Neurodegeneration, a BioMedCentral journal, titled RNAi mechanisms in Huntington’s disease therapy: siRNA versus shRNA.

After many years of development, RNAi therapeutics are nearing the clinic. There are several variants on RNAi therapeutics, such as antisense oligonucleotides (ASOs), short-hairpin RNA (shRNA), small interfering RNA (siRNA), et cetera. The researchers’ paper aimed to answer the question of why RNAi therapeutics for nucleotide repeat disorders (specifically Huntington’s, a devastating genetic neurodegenerative disease), have lost favor in recent years. After a phenomenal amount of excitement, these therapies were hindered by problems like molecular stability, dosing, and transcriptional control of the gene therapeutic construct.

“We compared various RNAi-based therapeutic modalities available for the treatment of Huntington’s Disease and offered mechanistic proposals on how to break through current barriers to clinical development. One key problem has proven to be modulating the expression level of shRNA constructs, which would otherwise be the clear frontrunner among ASOs, siRNAs, and other methods due to duration of expression, dramatically reduced off-target effects, and ease of delivery by adeno-associated viruses that are already approved by the EMA and FDA. We also put forward novel methods of modulating construct expression and avoiding off-target effects” said Franco Cortese, co-author of the paper and Deputy Director of the Biogerontology Research Foundation.

The researchers analyzed available data on the levels of off-target effects associated with siRNA vs shRNA, surveyed emerging strategies to reduce off-target effects in shRNA therapies (such as tough decoy RNAs, or TuDs), and proposed novel methods of controlling shRNA expression, in particular through the use of negative feedback-driven oscillating promoters.

Mechanism of TFEB at the PGC1-a promoter. The PGC1a promoter contains a CLEAR-box that is known to be bound by TFEB, a transcription factor induced during autophagy and lysosomal biogenesis. A construct being the PGC1a promoter CLEAR-box would be induced by TFEB under conditions of intracellular proteotoxicity due to HTT aggregation. By this mechanism, on-demand suppression of HTT could be achieved | Credit: Translational Neuroscience

 

“We proposed two novel feedback mechanisms that 1) activate construct expression stoichiometrically with mutant Huntingtin expression, or 2) only during aggregate-induced autophagy and lysosomal biogenesis. That way, the problem of excessive construct expression may be mitigated. These ideas were inspired by feedback systems used in synthetic biology, and in ‘nonsynthetic,’ naturally occurring biological systems” said Sebastian Aguiar, lead author of the paper.

Readers can read the open-access paper here: https://translationalneurodegeneration.biomedcentral.com/articles/10.1186/s40035-017-0101-9.

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About the Biogerontology Research Foundation

The Biogerontology Research Foundation is a UK non-profit research foundation and public policy center seeking to fill a gap within the research community, whereby the current scientific understanding of the ageing process is not yet being sufficiently exploited to produce effective medical interventions. The BGRF funds and conducts research which, building on the body of knowledge about how ageing happens, aims to develop biotechnological interventions to remediate the molecular and cellular deficits which accumulate with age and which underlie the ill-health of old age. Addressing ageing damage at this most fundamental level will provide an important opportunity to produce the effective, lasting treatments for the diseases and disabilities of ageing, required to improve quality of life in the elderly. The BGRF seeks to use the entire scope of modern biotechnology to attack the changes that take place in the course of ageing, and to address not just the symptoms of age-related diseases but also the mechanisms of those diseases.

About the Swammerdam Institute for Life Sciences

The Swammerdam Institute for Life Sciences (SILS) is the largest institute of the Faculty of Science at the University of Amsterdam. The institute comprises biological disciplines including molecular and cell biology, microbiology, plant science, physiology and neurobiology, supported by modern enabling technologies for the life sciences. The research groups of SILS also develop methods in genomics (micro-array, next-gen sequencing, proteomics), bioinformatics and advanced light microscopy technologies. Knowledge from adjacent fields of science, in particular biochemistry, biophysics, medicine, bioinformatics, statistics and information technology make SILS a multidisciplinary research institute with a systems biology approach to the life sciences. SILS’ research objective is to understand the functioning of living organisms, from the most basic aspects up to complex physiological function(s). Biological processes are studied at the level of molecules, cells, cellular networks and organisms. SILS research topics have in common that similar cellular processes and interactions are studied, likewise using similar methodologies and technologies. Therefore SILS scientists often study the same concepts in different biological systems. Within the institute, this leads to exchange of information and extension of research over the borders of different disciplines. Part of SILS research activities are directed to application-oriented research in close collaboration with industry.

Jim Mellon Announces Launch of New Book – Juvenescence: Investing in the Age of Longevity – Press Release by Biogerontology Research Foundation

Jim Mellon Announces Launch of New Book – Juvenescence: Investing in the Age of Longevity – Press Release by Biogerontology Research Foundation

Biogerontology Research Foundation


London, UK: Biogerontology Research Foundation Trustee Jim Mellon announces the publication of his newest book, Juvenescence: Investing in the Age of Longevity.

The book is a comprehensive summary of the emerging longevity industry, including profiles of longevity companies, investment opportunities, and aims to chart the major ideas of the geroscience’s thought-leaders and the vast implications this will have on economies and societies.

Often referred to as the British Warren Buffett, Billionaire Jim Mellon is well known for identifying major emerging trends before they become mainstream. Mellon made his wealth by investing in emerging markets throughout the 1990s. After many years of research and investing in the life science sector, Jim announced his vision for the emergence of the nascent longevity industry at Master Investor, one of the UK’s leading investor show in March of 2017, which was attended by over five thousand investors and entrepreneurs.

“The Biogerontology Research Foundation is proud to support what we feel will come to be seen as a landmark publication in the modern history of biogerontology. Jim has put an enormous amount of thought and effort into this new book, and has interviewed many of the field’s leading scientists in his research. The field of geroscience and the emerging longevity industry are both sure to prosper from very well-respected business personalities like Jim Mellon championing the longevity industry and projecting that it will become the world’s largest industry. Further, governments and policy makers should note the pressing need for a paradigm shift in medicine and healthcare away from ‘sick care’ toward comprehensive and disease-preventative healthspan extension. We are proud to have Jim as a Trustee of the Biogerontology Research Foundation and look forward to helping him lend mainstream credibility to the field and actionability to the dawning longevity industry” said Franco Cortese, Deputy Director & Trustee of the Biogerontology Research Foundation.

In 2012, Jim published his best-seller Cracking the Code, which summarized his vision for the future of the life science sector. In 2017, he announced his intention to focus much of his time and assets on the emerging longevity industry and make substantial investments into this area.

Jim and long-time co-author Al Chalabi toured many academic institutions, biopharmaceutical companies, and Silicon Valley tech companies to learn about the latest research and to understand the rapidly advancing field of longevity. Juvenescence highlights the technologies they deem the most likely to generate substantial longevity dividends and create sustainable and profitable industries. They travelled through the US and Europe, interviewing geroscience’s leading scientists and thought-leaders and provide an objective survey of their findings well as a detailed vision for the industry’s future and the most appropriate investment opportunities within the dawning longevity industry.

 

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Earlier this year Jim Mellon announced the formation of Juvenescence Limited, a company investing in the longevity biotechnology. Since then the company announced investments in several high-profile longevity companies including Insilico Medicine, Inc, a Baltimore-based leader in artificial intelligence for drug discovery, biomarker development, and aging research.

About the Biogerontology Research Foundation:

The Biogerontology Research Foundation is a UK non-profit research foundation and public policy center seeking to fill a gap within the research community, whereby the current scientific understanding of the ageing process is not yet being sufficiently exploited to produce effective medical interventions. The BGRF funds and conducts research which, building on the body of knowledge about how ageing happens, aims to develop biotechnological interventions to remediate the molecular and cellular deficits which accumulate with age and which underlie the ill-health of old age. Addressing ageing damage at this most fundamental level will provide an important opportunity to produce the effective, lasting treatments for the diseases and disabilities of ageing, required to improve quality of life in the elderly. The BGRF seeks to use the entire scope of modern biotechnology to attack the changes that take place in the course of ageing, and to address not just the symptoms of age-related diseases but also the mechanisms of those diseases.

Biogerontology Research Foundation Launches Campaign for Photographic Biomarkers of Age

Biogerontology Research Foundation Launches Campaign for Photographic Biomarkers of Age

Biogerontology Research Foundation


Thursday, August 31st, 2017, London, UK: The Biogerontology Research Foundation announces the launch of a crowdfunding campaign, MouseAge, to develop and test photographic biomarkers of ageing in mice in collaboration with scientists from Harvard University, University of Oxford, Youth Laboratories and Insilico Medicine. The project’s aim is to develop novel biomarkers of ageing in mice for the purposes of testing the effect of healthspan and lifespan-extending interventions. The project is now live at Lifespan.io, a crowdfunding platform for ageing research institutions that has launched several successful campaigns for SENS Research Foundation, International Longevity Alliance and CellAge.

“One of the most fundamental challenges in ageing research today is the development of robust and reliable biomarkers of ageing to serve as the basis by which the efficacy of lifespan and healthspan-extending interventions can be tested. Humans live a long time, and testing the effect of geroprotective interventions in humans using lifespan gains as the main criterion for success would be wildly impractical, necessitating long and costly longitudinal studies. By developing accurate biomarkers of ageing, the efficacy of potential geroprotective interventions could instead be tested according to changes in study participants’ biomarkers of ageing. While significant attention is paid to the development of highly accurate biomarkers of ageing, less attention is paid to developing actionable biomarkers of ageing that can be tested inexpensively using the tools at hand to the majority of researchers and clinicians. The Biogerontology Research Foundation chose to support MouseAge.org because photographic biomarkers of ageing represents a highly actionable alternative to more expensive measures of biological age.” said Franco Cortese, Deputy Director & Trustee of the Biogerongology Research Foundation.

The project utilizes Insilico Medicine’s novel deep learning platforms to correlate changes in physical appearance with biological and chronological age. Insilico is leading the pack in the intersection of deep learning and ageing research, and is well known for its use of advances in genomics, big data analysis, and deep learning for in silico drug discovery and drug repurposing for ageing and age-related diseases. The Biogerontology Research Foundation has collaborated with Insilico Medicine in the development of actionable and practical biomarkers of ageing before through their Ageing.AI project, and both organizations hope that MouseAge is the next in a long line of continuing research into the development of actionable biomarkers of ageing via the intersection of longevity research and deep learning.

“There are many experiments conducted around the world that examine lifespan in mice. The artificially intelligent MouseAge system will help determine which interventions make mice look younger. The plan is to develop an accurate predictor of mouse biological age based on images of mice and then apply transfer learning techniques to other datasets and data types,” said Vadim Gladyshev, MouseAge Research Lead and Professor of Medicine at Brigham and Women’s Hospital, Harvard Medical School.

Milestones for the project include the design of standardized protocols for creating photos and videos of mice, developing a mobile app and server infrastructure for image data collection, developing and testing the project’s main algorithm for mouse age prediction, optimizing feature extraction to investigate visual biomarkers of ageing in mice, creating a central data repository for the project’s data, utilizing transfer learning techniques to make these methods applicable to other model organisms, and ultimately using transfer learning techniques to develop photographic biomarkers of ageing in humans. The project’s principal investigator is Anastasia Georgievskaya, co-founder of Youth Laboratories, a company working at the intersection of ageing research, AI and machine vision, with the ultimate goal of using facial imageing data to predict patient health status.

The ultimate end-goal of MouseAge is to develop an intuitive mobile app to be used by researchers across the globe free of charge, where users can take images of model organisms and have the project’s DP-based algorithms perform age-assessment of images uploaded by users of the app. Both the organizations and researchers behind MouseAge are united in their belief in the promise of AI to accelerate ageing research and to streamline the development of effective healthspan-extending interventions for use in human patients, and hope that MouseAge comes to be remembered as an important landmark in the ongoing paradigm shift away from costly and inefficient sick-care and toward morbidity compression and effective healthspan extension for the benefit of all.

“Ageing research is the most altruistic cause that can generate billions of quality-adjusted life years over time and save the global economy. We are very happy to contribute to and support the MouseAge project. Our Young.AI system for tracking multiple biomarkers during human ageing is currently in the alpha stage and is launching in the fall. However, the biological relevance of many of the biomarkers and interventions is yet to be established, and the MouseAge project contributes to the body of fundamental science required to bridge AI and longevity research. Please support the MouseAge project on LifeSpan.io to contribute to this grand effort”, said Alex Zhavoronkov, PhD, Chief Science Officer of the Biogerontology Research Foundation.

About the Biogerontology Research Foundation:

The Biogerontology Research Foundation is a UK non-profit research foundation and public policy center seeking to fill a gap within the research community, whereby the current scientific understanding of the ageing process is not yet being sufficiently exploited to produce effective medical interventions. The BGRF funds and conducts research which, building on the body of knowledge about how ageing happens, aims to develop biotechnological interventions to remediate the molecular and cellular deficits which accumulate with age and which underlie the ill-health of old age. Addressing ageing damage at this most fundamental level will provide an important opportunity to produce the effective, lasting treatments for the diseases and disabilities of ageing, required to improve quality of life in the elderly. The BGRF seeks to use the entire scope of modern biotechnology to attack the changes that take place in the course of ageing, and to address not just the symptoms of age-related diseases but also the mechanisms of those diseases.

Induced Cell Turnover: A Proposed Modality for In Situ Tissue Regeneration and Repair – Press Release by Biogerontology Research Foundation

Induced Cell Turnover: A Proposed Modality for In Situ Tissue Regeneration and Repair – Press Release by Biogerontology Research Foundation

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Biogerontology Research Foundation


Scientists at the Biogerontology Research FoundationFeinberg School of Medicine at Northwestern University and Swammerdam Institute of Life Sciences at the University of Amsterdam have published a paper on a proposed method of in situ tissue regeneration called Induced Cell Turnover (ICT) in the journal Human Gene Therapy. The proposed therapeutic modality would aim to coordinate the targeted ablation of endogenous cells with the administration of minimally-differentiated, hPSC-derived cells in a gradual and multi-phasic manner so as to extrinsically mediate the turnover and replacement of whole tissues and organs with stem-cell derived cells.

“One of the major hurdles limiting traditional cell therapies is low levels of engraftment and retention, which is caused in part by cells only being able to engraft at locations of existing cell loss, and by the fact that many of those vacancies have already become occupied by ECM and fibroblasts (i.e. scar tissue) by the time the cells are administered, long after the actual occurrence of cell loss. The crux underlying ICT is to coordinate endogenous cell ablation (i.e. induced apoptosis) with replacement cell administration so as to manually vacate niches for new cells to engraft, coordinating these two events in space and time so as to minimize the ability for sites of cell loss to become occupied by ECM and fibroblasts. This would be done in a gradual and multi-phasic manner so as to avoid acute tissue failure resulting from the transient absence of too many cells at any one time. While the notion of endogenous cell clearance prior to replacement cell administration has become routine for bone marrow transplants, it isn’t really on the horizon of researchers and clinicians working with solid tissues, and this is something we’d like to change,” said Franco Cortese, Deputy Director and Trustee of the Biogerontology Research Foundation, and lead author on the paper.

Cell-type and tissue-specific rates of induced turnover could be achieved using cell-type specific pro-apoptotic small molecule cocktails, peptide mimetics, and/or tissue-tropic AAV-delivered suicide genes driven by cell-type specific promoters. Because these sites of ablation would still be “fresh” when replacement cells are administered, the presumption is that the patterns of ablation will make administered cells more likely to engraft where they should, in freshly vacated niches where the signals promoting cell migration and engraftment are still active. By varying the dose of cell-type targeted ablative agents, cell type and tissue-specific rates of induced turnover could be achieved, allowing for the rate and spatial distribution of turnover to be tuned to the size of the tissue in order to avoid ablating too many cells at once and inadvertently inducing acute tissue failure.

“Cell therapies are limited by low levels of engraftment, and in principle their ability to improve clinical outcomes is limited by the fact that they can only engraft at locations of existing cell loss. Conversely, therapeutic tissue and organ engineering requires surgery, is more likely to introduce biochemical and mechanical abnormalities to tissue ultrastructure through the decellularization process, and is fundamentally incapable of replacing distributed tissues and structures with a high degree of interconnectivity to other tissues in the body. The aim of ICT is to form a bridge between these two main pillars of regenerative medicine, extending the efficacy of cell therapies beyond a patch for existing cell loss and accomplishing the aim of tissue and organ engineering (i.e. the replacement and regeneration of whole tissues and organs) while potentially remaining free of some of their present limitations,” said Giovanni Santostasi, co-author on the paper and a researcher at the Feinberg School of Medicine, Northwestern University.

While future iterations of the therapy could use patient-derived cells, such as ESCs derived via somatic cell nuclear transfer (SCNT) or iPSCs derived from nuclear reprogramming, shorter-term applications would likely use existing stem cell lines immunologically matched to the patient via HLA matching. The authors contend that the cloning of adult organisms with normal lifespans from adult somatic cells testifies to the fact that adult cells can be rejuvenated and used to produce a sufficient quantity of daughter cells to replace the sum of cells constituting adult organisms, and that serial cloning experiments (in which this process is done iteratively, using an adult cell of each subsequent generation to derive the next) attests to this fact even more strongly.

“ICT could theoretically enable the controlled turnover and rejuvenation of aged tissues. The technique is particularly applicable to tissues that are not amenable to growth ex vivo and implantation (as with solid organs) – such as the vascular, lymphatic, and nervous systems. The method relies upon targeted ablation of old, damaged and/or senescent cells, coupled with a titrated replacement with patient-derived semi-differentiated stem and progenitor cells. By gradually replacing the old cells with new cells, entire tissues can be replaced in situ. The body naturally turns over tissues, but not all tissues and perhaps not optimally. I am reminded of the quote attributed to Heraclitus: ‘No man ever steps in the same river twice, for it’s not the same river and he’s not the same man,'” said Sebastian Aguiar, a coauthor on the paper and researcher at the Swammerdam Institute of Life Sciences, University of Amsterdam.

“Reversing aging in humans will require a multi-step approach at multiple levels of the organismal organization. In situ targeted ablation of the senescent cells and regeneration will be an important component of comprehensive anti-aging therapies,” said Alex Zhavoronkov, Chief Science Officer of the Biogerontology Research Foundation.

The researchers originally proposed ICT in 2016 in the context of biomedical gerontology as a possible means of preventing and/or negating age-related phenotypic deviation for the purposes of healthspan extension, and in this new paper they refine the methodological underpinnings of the approach, take a closer look at potential complications and strategies for their deterrence, and analyze ICT in the context of regenerative medicine as an intervention for a broader range of conditions based on disease or dysfunction at the cellular and intercellular level, with potential utilities absent from traditional cell therapies and tissue/organ engineering, the two main pillars of regenerative medicine. The intervention is still very much conceptual, and any potential utilities over other therapeutic modalities within regenerative medicine would need to be verified via preclinical studies, but their hope is to stimulate further research at this interface between geroscience and regenerative medicine.

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The paper is available here.

About the Biogerontology Research Foundation:

The Biogerontology Research Foundation is a UK non-profit research foundation and public policy center seeking to fill a gap within the research community, whereby the current scientific understanding of the ageing process is not yet being sufficiently exploited to produce effective medical interventions. The BGRF funds and conducts research which, building on the body of knowledge about how ageing happens, aims to develop biotechnological interventions to remediate the molecular and cellular deficits which accumulate with age and which underlie the ill-health of old age. Addressing ageing damage at this most fundamental level will provide an important opportunity to produce the effective, lasting treatments for the diseases and disabilities of ageing, required to improve quality of life in the elderly. The BGRF seeks to use the entire scope of modern biotechnology to attack the changes that take place in the course of ageing, and to address not just the symptoms of age-related diseases but also the mechanisms of those diseases.

Benchmark Database of Lifespan-Extending Drugs Announced – Press Release by Biogerontology Research Foundation

Benchmark Database of Lifespan-Extending Drugs Announced – Press Release by Biogerontology Research Foundation

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Note from the Editor: The following Press Release was originally published on EurekAlert! Science News by the Biogerontology Research Foundation. In accord with the U.S. Transhumanist Party’s open support of longevity science – in particular, Article V and Article VI of the Transhumanist Bill of Rights (v. 2.0) – we affirm our support by republishing the Press Release below.

~ B.J. Murphy, Director of Social Media, United States Transhumanist Party, March 16, 2017


Benchmark database of lifespan-extending drugs announced

Finding: The majority of age-related pathways have yet to be targeted pharmacologically

BIOGERONTOLOGY RESEARCH FOUNDATION

Scientists from the Biogerontology Research Foundation (BGRF) and University of Liverpool have announced a landmark database of lifespan-extending drugs and compounds called DrugAge. The database has 418 compounds, curated from studies spanning 27 different model organisms including yeast, worms, flies and mice. It is the largest such database in the world at this time. Significantly, the study found that the majority of age-related pathways have not yet been targeted pharmacologically, and that the pharmacological modulation of aging has by and large focused upon a small subset of currently-known age-related pathways. This suggests that there is still plenty of scope for the discovery of new lifespan-extending and healthspan-extending compounds.

DrugAge is the latest of a number of valuable resources freely available on the Human Aging Genomic Resources (HAGR) website created and maintained by the Integrative Genomics of Ageing Group at the University of Liverpool, led by Biogerontology Research Foundation Trustee Joao Pedro de Magalhaes, in collaboration with many other scientists worldwide, including BGRF Chief Science Officer and CEO of Insilico Medicine, Alex Zhavoronkov, PhD. Other resources available through HAGR include GenAge (a database of age and longevity-related genes in humans and model organisms), AnAge (a database on ageing, longevity records and life-history featuring over 4000 species), GenDR (a database of genes associated with the life extending effects of dietary restriction), and LongevityMap (a database of over 2000 human genes and genetic variations associated with longevity).

“DrugAge is the latest database created by Joao Pedro de Magalhaes, a world leader in the application of advanced bioinformatics and integrated computational approaches to biogerontology and ageing research. I am confident that it will gain widespread use in the ageing research community, and represents a significant milestone along the way to the coming paradigm shift in modern healthcare away from single disease treatment and toward geroprotective multi-disease prevention,” said Dmitry Kaminskiy, Managing Trustee of the Biogerontology Research Foundation.

The database is freely available to the public, and is searchable according to compound name, species and effect on lifespan. The data can be presented as both tables and interactive charts. Functional enrichment analysis of the targets of the database’s compounds was performed using drug-gene interaction data, which revealed a modest but statistically significant correlation between the cellular targets of the database’s compounds and known age-related genes.

The database encompasses the earlier efforts published by the BGRF scientists, Joao Pedro de Magalhaes, PhD and Alex Zhavoronkov, PhD as an open resource called Geroprotectors.org. The publication is available at http://www.aging-us.com/article/100799 .

“DrugAge represents a landmark resource for use in the biogerontology community. It is the largest database of lifespan-extending compounds compiled to date, and will surely come to be recognized as an extremely valuable resource for biogerontologists. Analysis performed using the database has already revealed interesting trends, including a modest but statistically significant overlap between lifespan-extending drugs and known age-related genes, a strong correlation between average/median lifespan changes and maximum lifespan changes, a strong correlation between the lifespan-extending effects of compounds between males and females, and perhaps most significantly that most known age-related pathways have yet to be targeted pharmacologically. More broadly, an understanding of the comparative effects of geroprotectors upon the lifespan of a variety of different model organisms is important both for basic research into the biology of ageing, demonstration of lifespan plasticity via modulation of a variety of distinct biomolecular targets as proof to regulators that healthspan extension is a viable paradigm for disease treatment and prevention, and for the eventual clinical translation of potential geroprotectors,” said Franco Cortese, Deputy Director and Trustee of the Biogerontology Research Foundation.

“Besides introducing the DrugAge database to the larger scientific community, this paper’s overarching significance lies in the finding that the large majority of known age-related pathways have not yet been targeted pharmacologically, and that we are in a very real sense at the starting line of the search for pharmacological agents capable of extending lifespan and healthspan via the modulation of known age-related pathways. There is still very much left to learn,” said Joao Pedro de Magalhaes, PhD, a Trustee of the Biogerontology Research Foundation (BGRF) and a Principal Investigator at the University of Liverpool’s Integrative Genomics of Aging Group (IGAG).

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The paper ‘The DrugAge database of ageing-related drugs’ has been published in the journal Aging Cell.