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To date, there are no curative treatments for most neurodegenerative disorders, including synucleinopathies of the central nervous system (CNS) such as Parkinsons disease (PD). The greatest challenges in treating these are currently insufficient penetration of drugs to the parenchyma of the brain and achieving stable reduction of α-synuclein (SNCA) protein aggregation, the most significant hallmark of PD pathology. Therefore, it is imperative to find new solutions to these unmet ̽»¨¾«Ñ¡ needs. Recently, we have harnessed nature’s own nanoparticles, extracellular vesicles (EVs), for targeted delivery of biotherpeutics, with focus on Cas9-based gene editors. Here, we will establish a novel CNS delivery platform exploiting these engineered EVs for CRISPR/Cas9-mediated silencing of SNCA gene expression and prevention of α-synuclein aggregation. After optimization of EV engineering components focusing on enhanced cargo loading, gene editing, and EV delivery to deep brain parenchyma, we will demonstrate their therapeutic effect in models of PD by local injection into the brain of mice using osmotic pumps. If successful, this will open an entirely new avenue to treat PD and other synucleinopathies. Importantly, the modular nature of the EV engineering platform permits further adaptation to deliver other gene editors or drug modalities, thereby opening innovative possibilities to treat a range of CNS diseases.

Genome-targeted therapies, e.g., CRISPR-Cas9, use genetic sequence to target their actions to specific genes, bringing unrivalled specificity. This specificity will revolutionise healthcare, bringing therapies to previously untreatable rare disorders and precision gene- and cell-specific approaches to common disorders. By simply modifying the target sequence, a therapy can be retargeted to treat a new disorder. Our challenge is to unlock this potential, by developing 'Therapeutic Genomics' (TG), a genome-led, patient-centred, data-driven vision to bring genome-targeted therapies to the clinic, at scale. To achieve this challenge, our research aims to answer five major questions. First, which variants and which patients can be treated? We will develop computational and experimental methods to identify tractable genetic variants with different genome-targeted therapies. Applying these to genome sequencing data collected nationwide by researchers and the NHS, we will identify patients with correctable variants and symptoms appropriate to experimental therapies. Second, how can Therapeutic Platforms be developed and retargeted at scale to treat numerous genetic disorders? In collaboration with the laboratory that discovered CRISPR-Cas9 (UC Berkeley), we will develop exemplar Therapeutic Platforms and then retarget these to develop numerous therapies for other variants and disorders. Working with our industry partners, including Intellia and Danaher, we will develop high-throughput parallelized methods for measuring potency and safety in scalable cellular models, reducing the reliance on animal testing. Subsequent generations of Therapeutic Platforms and retargeting will extend therapies to more disorders. Third, how can Therapeutic Platform therapies be delivered effectively? At present, we lack a suitable vector for targeting neurons throughout the brain, a hurdle complicated by the inaccessibility of mature human neurons. Using cutting-edge technology to maintain human neuronal viability post-mortem, we will use directed evolution to develop brain-wide and cell-type-specific delivery systems, including viral, non-viral, and hybrid approaches. Fourth, how can insights from one therapy be used to improve the next therapy? By collecting data throughout our CoRE's research, we will apply artificial intelligence to refine our therapeutic and delivery technologies, ultimately developing safer and more effective therapies that treat an ever-expanding list of disorders. The data and algorithms from this work will be shared publicly to help design suitable therapies for all patients who need them. Fifth, how can we foster the research culture and clinical ecosystem to bring therapies to the clinic? Our research aims can only be achieved through a positive, inclusive research culture (e.g., openness, collaboration, teamwork, integrity, and diversity), and an interdisciplinary team. The MRC CoRE in TG will build on institutional initiatives to support culture change, embracing opportunities to enhance mentorship, teamwork, and communication. Key principles of Equality, Diversity, and Inclusion (EDI) and inclusive Public Patient Involvement and Engagement (PPIE) will be fully integrated into, and will enrich, the planning and execution of the CoRE's activities and governance. The MRC CoRE in TG will prime the clinical landscape required to bring genome-targeted therapies to the clinic, in collaboration with stakeholders and patients. Our team has the experience and facilities to secure additional funding and conduct first-in-human clinical trials. We will enlist UK infrastructure and industrial collaboration to reduce manufacturing costs and engage with the UK and other regulatory agencies to define workable standards for assessing Therapeutic Platforms. By developing urgently needed therapies for multiple rare disorders today, we will develop the expertise and capacity to better treat all rare and common disorders in the future.

Nucleic acid therapies (NATs) are genetic medicines that address the root cause of disease and have the potential to transform healthcare and provide life changing solutions for numerous areas of unmet need. Neurological, neuromuscular and cardiovascular diseases in particular devastate lives and create a very significant economic and social burden across the entire global population. While NATs have begun to be a reality over the last decade with multiple medicines being approved for use in the US and Europe many challenges remain particularly for diseases outside the liver and for those not easily addressed by local drug delivery solutions. Moreover, recent clinical trial results indicate that safety considerations should be addressed in parallel with the development of delivery solutions. The challenge of NAT delivery put simply is to deliver the drug effectively across the cell membrane into the appropriate sub-cellular compartment at a sufficient concentration required for activity in the absence of significant safety signals - so called 'productive' delivery. Our proposed solution is therefore to understand the requirements for productive delivery of NATs and to exploit this knowledge base for the development of NAT conjugates - our technical solution. Building on extensive experience of our consortium of academic and industry scientists, we will take two independent approaches to NAT conjugates, where delivery agents are directly chemically attached to the NAT drug. First, we will study and optimise lipid conjugates, where a range of lipid entities are directly attached to the NAT via a series of chemical linkers with different properties. In the first instance the NAT is one targeting a common gene of no therapeutic relevance. Our second approach of high potential will be to study and optimise antibody conjugates, where an antibody (or antibody fragment or antibody derived peptide) that binds to a specific cell membrane ligand is conjugated chemically again via chemical linkers. In each case, we will have starting points with conjugates that have already emerged through the work of consortium members, and in the case of antibodies we will have two independent approaches for identifying and prioritising new ligands for antibody targeting, again building on pre-existing work in the consortium. Our extensive chemistry capabilities will generate conjugate materials and control compounds for study and first step of which will be extensive in vitro studies in cells to develop mechanism-based knowledge on productive cell uptake allowing us to select lead compounds for more detailed study based on cell uptake/efficacy/safety properties, and to iterate compound structure and chemistry based on new knowledge, know-how and data generated. Further study will comprise translational studies in ex vivo human model systems based on human cells and stem cells and also based on human three dimensional organ like systems that provide cell diversity and architectural arrangements more closely mimicking human tissues. Further translational studies in established and new rodent models will allow delivery to cells and tissues of brain, heart and muscle to be studied in detail at singe cell resolution permitting cell/tissue biodistribution to be correlated with efficacy and safety measures. Finally, a small number of lead NAT compounds will be studied in disease models related to Huntington's disease and muscular dystrophy. We will maximise the potential of data by analysing and integrating across the programme and implementing machine-learning approaches to exploit our data. We will deliver fundamental knowledge, know-how, data and IP on productive uptake and novel lipid/antibody NATs of high therapeutic potential for further study. We will also engage the broader NAT community via reports/meetings/conferences and develop training opportunities, all of the above working in close collaboration with the NATA Hub. Technical Summary CNS and neuromuscular diseases devastate lives and create vast global economic costs (>$817Bn in 2021). Nucleic acid therapeutics (NAT) have huge potential to treat such disorders by addressing the disease cause. Despite recent advances, significant challenges in NAT delivery to many cell/tissue types remain. Recent clinical trials have identified toxicity issues, emphasising the need to consider safety in parallel with efforts to improve delivery. Our aim is to achieve productive, safe NAT delivery, leading to the intended activity, and this can only be accomplished by advancing mechanism-based understanding and developing NAT bioconjugates that address the technical demands. Here we establish a world-leading consortium to address the NAT delivery challenge, advancing the study of improved NAT compounds to understand the mechanistic basis of productive NAT uptake and its implications for intracellular/in vivo delivery, trafficking, safety, and delivery to CNS, heart, and muscle. We will develop and study a range of next-generation NAT compounds (focussing on lipid and antibody/peptide bioconjugates) to be evaluated as follows: 1 Mechanistic in vitro studies of cell uptake, endosomal escape, compartment (cytoplasm/nucleus/mitochondria) localisation - quantifying productive cell uptake, distribution, safety and efficacy readouts. 2 Development of novel ex vivo human tissue organoid models to screen and evaluate cell/tissue delivery, safety, and efficacy. 3 Translational in vivo studies in wild-type and novel reporter rodent species to investigate CNS and heart/muscle delivery, using available consortium tool compounds against reporter/other targets e.g. MALAT1 and quantifying distribution and efficacy at single cell resolution. 4 Selection of lead NAT compounds to study in clinically relevant disease models e.g. HD and DMD. 5 All data will be analysed and integrated computationally to allow machine-learning-based advances in NAT development to be made.

Nucleic acid-based medicines have opened a new avenue in drug discovery to target currently undruggable genes and to express therapeutic proteins, unlocking novel therapeutic options for a range of diseases, including neurodegeneration. However, they need to be encapsulated in nanocarriers to ensure their stability and efficient uptake into cells and tissues. Synthetic nanoparticles based on cell-penetrating peptides (CPPs) and, particularly, lipid nanoparticles (LNPs) have recently emerged as potent vectors for hepatic delivery. However, these systems fail to robustly target other organs in a safe manner. Another promising nanocarrier for advanced drug delivery is extracellular vesicles (EVs) that have the ability to efficiently convey macromolecules into cells. As native nanoparticles, EVs benefit from immune tolerance as well as the ability to cross biological barriers to reach, for example, the brain. We have developed advanced strategies to bioengineer cells to generate EVs loaded with therapeutic RNAs and proteins. However, their production at scale is cumbersome and time consuming. Here, I propose a platform development using synthetic nanocarriers to transiently engineer hepatic cells in vivo and harness EVs to functionally DELIVER biotherapeutics to currently unreachable, distant organs, focusing on brain. To achieve this, genetic constructs will be developed that allow for transient in situ engineering of cells in vivo and release of cargo (e.g. CRE)- laden EVs, displaying CNS-specific peptides, that can be functionally transported to distant organs, including brain. We will exploit the same strategy using CPP-based nanoformulations, recently developed in my lab, injected locally in brain to secrete EVs loaded with the disease-relevant protein GBA1 as a treatment strategy for Parkinson´s disease. Long-term this novel project has enormous potential, as any engineered EV could be produced in situ and be used for delivery of virtually any biotherapeutics.