University College London, UK

Bar-Ilan University, Israel

Associate Professor | Head of Department, The Cyprus Institute of Neurology & Genetics

Dr Lederer received his PhD from the University of East Anglia for work in plant virology at the John Innes Centre, Norwich, UK. He is now Senior Scientist and Head of the Molecular Genetics Thalassaemia Department (MGTD) at the Cyprus Institute of Neurology & Genetics (CING), where he also leads the MGTD Gene Editing and Therapy unit. Dr Lederer is Associate Professor and course coordinator for CING Postgraduate Education, executive board member of the Cypriot National Committee for Thalassemia and the Global Globin Network, member of the core-development team of the ITHANET Portal, member of the ClinGen-recognised Hemoglobinopathies Variant Curation Expert Panel and member of the Cyprus Society of Human Genetics, the Hellenic Society of Gene Therapy and Regenerative Medicine, and the American Society of Gene & Cell Therapy.

Presentation Abstract

Current gene editing tools and trends – an overview

Gene editing is revolutionizing therapy development and biomedical research. Mostly based on elements of three main editing platforms, the field has experienced a major impetus in take-up, breadth of application and diversity by distribution, optimization, modification and modular extension of the versatile and wieldy CRISPR/Cas system. The resulting and ever-increasing plethora of editing tools comprises classical genome editors based on DNA double-strand breaks (DSB) and DSB-independent DNA and epigenome editors, which in turn find application by a range of ex vivo and in vivo delivery strategies to a growing number of human diseases. Toward clinical application, research focusses on fine-tuning cell-type and on-target specificity, efficiency, precision and tolerability of editing, as well as on the development of associated analysis technology.
This talk will give an overview of corresponding trends and achievements in the field of gene editing, introductory to concepts of relevance for other talks during this conference and with emphasis on aspects critical to therapy development for human diseases.

 Senior Research Fellow, UCL Great Ormond Street Institute of Child Health

Dr. Georgiadis, is an MRC/AstraZeneca trust funded senior postdoctoral research fellow based in the Molecular and Cellular Immunology section at the Great Ormond Street Institute of Child Health, UCL. Over the past 12 years he has been researching gene therapy strategies for the treatment of neurodegenerative, cutaneous and haematological diseases.

In his current role, Christos has been operating at the translational interface between laboratory research and clinical application alongside Prof. Waseem Qasim. In this position, he leads a small team of talented PhD students and research scientists focusing on the use of novel scarless base editing technologies for the development of readily available therapies catering for the unmet clinical need. Such proof-of-concept studies have led to world-first Phase 1 testing of universal, ‘off-the-shelf’ CAR T cell therapies for paediatric B and T cell acute lymphoblastic leukaemias at GOSH, with work currently underway aiming to broaden applicability to a wider group of genetic disorders.

Beyond his research endeavours, Christos leads the Public Engagement committee for the British Society for Gene and Cell Therapy. In this capacity, he has contributed to improving public awareness and promoting responsible and informed science through free webinars and science days for various patient focus groups and early career researchers.

Presentation Abstract

Base-Edited ‘universal’ CAR7 T cells for T-cell malignancies

Genome editing has successfully been used to overcome HLA barriers and generate 'off-the-shelf’ chimeric antigen receptor (CAR) T cell therapies for B-cell malignancies. CRISPR-Cas9 has previously been employed for the generation of ‘universal’ T cells devoid of TCRαβ and CD52 expression. However, multiplexed nuclease-mediated genome editing can result in double stand DNA (dsDNA) breaks, and an increased risk of chromosomal translocations.
CRISPR-guided cytidine deamination mediates highly precise conversion of C>T which can be used to directly disrupt gene expression across multiple sites without DNA breaks mitigating the risk of translocations and other chromosomal aberrations.
We generated “universal”, “off-the-shelf” CAR T cell (BE-CAR7) investigational medicinal products (IMPs) using a semi-automated process from steady state apheresis harvests from allogeneic healthy donors under compliant conditions. Using codon-optimised base-editor(coBE) mRNA we inactivated three genes encoding CD7, CD52 receptors and the T-cell receptor αβ chain to prevent CAR7-T cell fratricide, graft rejection, and graft versus host disease, respectively, and transduced these with a lentivirus to express a CAR against CD7 (CAR7), a protein expressed by T cell acute lymphoblastic leukaemia (T-ALL).
A Phase 1 clinical trial sponsored by Great Ormond Street Hospital will investigate feasibility and safety of base edited allogeneic CAR T cells in R/R paediatric T-ALL. The first patient, aged 13, who had relapsed T-ALL after allogeneic stem cell transplantation (allo-SCT), achieved molecular remission within 28 days of infusion of a single dose of BE-CAR7 prior to receiving a reduced-intensity (non-myeloblative) allo-SCT-stem cell transplant, with successful immunological reconstitution and ongoing leukaemic remission.
The study has demonstrated the feasibility of "off-the-shelf” base edited CAR7 T cells supporting their further investigation for treatment of patients with relapsed T-ALL.

PhD, Medical Center - University of Freiburg

Dr. Claudio Mussolino is group leader at the Institute for Transfusion Medicine and Gene Therapy at the University Medical Center Freiburg, (Germany). His research focuses on the establishment of novel strategies to edit the genome or the epigenome of hematopoietic cells. Using TALE- or CRISPR-based effectors, Dr. Mussolino explores their use for the treatment of chronic and acquired immunodeficiencies as well as cancer. His group has pioneered the development of epigenome editors highlighting the potential of this technology to establish novel therapeutics to fight infection with the human immunodeficiency virus (HIV). Additional current projects include the adaptation of these methodologies to render CAR T cells resistant to tumor-induced exhaustion.

Presentation Abstract

Mitigation of on target mutagenesis with HDR-CRISPR

Genome editing using designer nucleases promises to revolutionize modern medicine as it allows the direct correction of mutations causing devastating genetic disorders. However, safety concerns remain as a consequence of the unwanted modifications that arise from designer nucleases activity. I will discuss of our efforts in promoting seamless genome editing using modified CRISPR-based nucleases. HDR-CRISPR consists of a Cas9 fused to DNA repair factors to synergistically inhibit NHEJ and favor HDR for precise repairing of Cas-induced DSBs. Use of HDR-CRISPR results in a significant increase in error-free editing ranging from 1.5-fold to 7-fold in multiple cell lines and in primary human cells. These results provide a remarkable gain in safety and advocates this novel CRISPR system as an attractive tool for therapeutic applications depending on precision genome editing.

Oslo University, Norway

Miltenyi Biotec, Germany

Technical University Dresden, Germany

Frank is Professor of Medical Systems Biology and Head of Translational Research at the University Cancer Center (UCC) at TU Dresden since 2010. He has been Dean of Research at the Carl Gustav Carus Faculty of Medicine since 2022. Already during his doctorate (1994 - 1998), he did groundbreaking work on the implementation and improvement of sequence-specific recombinases in the field of genome engineering. Frank perfected this approach as group leader at the MPI-CBG Dresden (2002 - 2010) and developed the Tre recombinase, an enzyme that can eliminate HIV from infected cells. This completely novel antiretroviral approach was widely recognized as a breakthrough in enzyme technology and offers a new strategy to cure patients with HIV infection. Based on these results, he co-founded Provirex Genome Editing Therapies GmbH together with colleagues from Hamburg in 2019. With the help of BMBF GO-Bio funding, the recombinase platform was further developed, which led to another TUD spin-off in 2022. Seamless Therapeutics GmbH was founded at the beginning of 2023. This makes Frank Buchholz one of Germany's most active academic founders in the life science sector, making an important contribution to bringing the latest therapies to patients as quickly as possible.

Presentation Abstract

Engineering Designer-Recombinases for therapeutic genome editing

Recombinases have several potential advantages as genome editing tools compared with nucleases and other genome editing enzymes, but efforts to engineer them to bind specific DNA targets of interest are time consuming and labor intensive. We have overcome many of the limitations of reprogramming recombinases to therapeutically relevant sites. I will present our platform of designer-recombinase engineering, including the latest advancements in programming recombinases through zinc-finger fusions.

Professor, Group leader, University of Copenhagen

Jan Gorodkin holds a MSc in physics from the Niels Bohr Institute and obtained his Ph.D. in Bioinformatics from Center for Biological Sequence analysis at the Technical University, Denmark. With outset in an awarded talent project grant from the national research council, he did a joint post doc at Aarhus University, Denmark and Washington University Medical School, St. Louis. He has since then been awarded numerous grants. He took up positions at the Royal Veterinary and Agricultural University (now University of Copenhagen), where he was also director of Center for non-coding RNA in Technology and Health and became professor and head of the bioinformatics group in the Department of Veterinary and Animal Sciences. His research interests span from RNA and CRISPR bioinformatics to genome analysis. His is research group has been involved in developing numerous computational tools as well as applying them on genomic and transcription data.

Lab website
https://ivh.ku.dk/bioinformatics
https://rth.dk

Presentation Abstract

Computational CRISPR gRNA design: From Cas9 to base editing and Cas12a

CRISPR gRNA design is essentially a matter of scoring all relevant gRNA possibilities within the context a specific intended genome edit. Building on the CRISPRon method we have developed a state-of-the-art base editing gRNA design tool, CRISPRon-BE for Adenosine and Cystosine base editing (ABE/ CBE) respectively. We generated efficiency data for ~11,500 novel gRNAs for both ABE and CBE and combined these with publicly available data obtaining efficiencies for ~17,000 gRNAs for both. After data cleaning approximately half the data points can meaningfully be used to construct CRISPRon-ABE and CRISPRon-CBE respectively. These methods are simultaneous trained on the different available data sets of which the performances of the individual data sets mutually benefit from mutual training. Upon testing CRISPRon-A/CBE and exiting methods we on various combinations of independent test sets obtain performance improvements in rank correlation coefficients with e.g. for ABE from ~0.77 to ~0.85. For Cas12a we change the computational method design to incorporate a secondary-structure-enhanced attention module made by adding the features extracted from the gRNA base pair probabilities and the positional distances in the gRNA sequence. This is implemented a as self-attention layer similar to that in a Transformer. Making using of ~15,000 gRNA publicly available efficiencies, we which downsample to ~12,000 to ensure a flat distribution we obtain on three independent data sets obtain improvement over current methods in rank correlation of 2.7, 4.7, and 1.0 with performances 79.2, 79.2, and 58.8, respectively.

GENYO, Spain

Vice president Gene Therapy, Cellectis

Dr. Julien Valton obtained his Ph.D. at the Université Grenoble Alpes in Grenoble, France, where he was trained as an enzymologist/biophysicist. He then joined the Yale University School of Medicine to apply his knowledge to therapeutic research by investigating the mechanism of inhibition of receptor tyrosine kinases that are involved in the development of gastrointestinal cancer. In 2009, he moved a step further into the field of applied science by joining the Innovation Department of Cellectis, where he actively participated in setting up, improving, and using the TALEN® gene editing technology for targeted gene therapy and genome engineering. He used TALEN® along with protein engineering techniques to develop SMART CAR T-cells that sense and adapt to tumor microenvironment to address different malignancies. He is now applying his knowledge to develop the next generation of Cellectis’ gene therapy products to treat different genetic diseases including Sickle Cell Anemia and Inborn Metabolic Diseases.

Presentation Abstract

Non-viral DNA delivery coupled to TALEN gene editing efficiently corrects the sickle cell mutation in long-term HSCs

Sickle cell disease stems from a single point mutation in the HBB gene that results in sickle hemoglobin. Nuclease-based gene therapy approaches provide a therapeutic route for restoring functional hemoglobin in sickle cell patients. In this report, we leveraged TALEN technology to develop a gene editing process that enables highly efficient HBB gene correction via homology-directed repair while mitigating potential risks associated with HBB gene knock-out. To achieve this, we compared viral (AAV6) versus non-viral (ssODN) DNA template delivery in clinically relevant hematopoietic stem and progenitor cells (HSPCs). Both strategies led to comparable high efficiency HBB gene correction in vitro in healthy donor (HD) and homozygous sickle patient (HbSS) HSPCs, without affecting their viability, purity, or clonogenic potential. Moreover, both strategies elicited more than 50% expression of normal adult hemoglobin in red blood cells without inducing β-thalassemic phenotype or major transcriptional changes. In an in vivo immunodeficient mouse model, transplanted ssODN-edited HSPCs showed higher levels of engraftment and gene correction than did AAV6-edited HSPCs from HDs or HbSS patients. Further characterization of edited HSPCs by single-cell transcriptomics revealed that ssODN-based editing led to lower p53 activation and the maintenance of higher proportions of primitive HSCs in HD and HbSS than did AAV6-based editing. Therefore, non-viral DNA delivery coupled with TALEN gene editing reduces toxicity linked to viral DNA delivery and allows greater HBB gene correction in long-term hematopoietic stem cells (LT-HSCs). These results provide the basis for the development of an effective TALEN-based autologous gene therapy for sickle cell disease.

University of Granada, Spain

Visiting Professor, University College London, UK

I am Vice the President of Translational Science at Nvelop Therapeutics and a Visiting Professor at University College of London (UCL).
I pioneered the field of clonal tracking of genetically engineered cells in humans, with a focus on the characterization and in vivo engineering of human hematopoietic/stem progenitor cells. As academic and industry research lead, I have authored several publications in high-impact scientific journals such as Science, Nature Medicine, Nature Cancer and Cell Stem Cell and I have been the recipient of 5 major international awards in Gene Therapy and Hematology.

I graduated in Medical Biotechnology in 2003 at Alma Mater Studiorum University of Bologna, Italy and moved to Milan to join the San Raffaele Telethon Institute of Gene Therapy (SR-TIGET) where I obtained my PhD in 2010 and became later a group leader specializing in the study of retroviral/lentiviral vector-host interactions. In 2016 I moved to Boston MA, US as Assistant Professor at Harvard Medical School where I directed the vector safety team for the Gene Therapy program at Boston Children's Hospital/Dana Farber Cancer Institute. My research laboratory worked on understanding the properties of stem cell gene therapy products and their in vivo dynamics upon infusion in humans. Over the same period, I was appointed Senior Research Associate and then Visiting Professor at UCL, in London UK where I am conducting translational research on immunodeficiencies and CAR- T cells. In 2019 I joined AVROBIO a gene therapy company based in Cambridge MA, US where I applied state-of-the-art single cell molecular analyses to track the fate and activity of genetically engineered cells in humans. Since 2022 I am leading R&D teams working on the in vivo delivery of genetic payloads to hematopoietic stem cells and other relevant cell types at SANA biotechnology first and now at Nvelop Therapeutics.

Presentation Abstract

Efficient and specific in vivo genetic engineering of human hematopoietic stem/progenitor cells

Achieving in vivo genetic engineering of hematopoietic stem progenitor cells (HSPC) has the potential to transform treatment for hematological disorders. An in vivo delivery platform should provide access to resting HSPC at low doses, in different in vivo compartments and with high specificity. We developed a multi-model testing strategy and achieved efficient and specific genetic engineering of human HSPC using potent lentiviral vectors (LV) and viral-like particles (VLP). To model access to HSPC in the peripheral blood (PB), we followed long-term NBSGW mice where BaEVTR-LV was dosed intravenously (IV) immediately after HSPC infusion, showing targeting of 23% early-engrafting bone marrow (BM) HSPC and stable 5-7% engineered myeloid chimerism at wk16. In a separate study we instead dosed BaEVTR-LV at D7 post-humanization, a timepoint when we established that engrafted human HSPC have physiological composition and are confined to the BM, formally demonstrating access to 5% BM-resident HSPC. Based on these data, we developed a potent BaEVTR VLP carrying a CRISPR/Cas9 editor targeting the B2M locus. This VLP displayed 92.5% editing efficiency in vitro in resting HSPCs. When dosed in vivo in the D7 post-humanization model, our VLP up to 31% B2M KO in BM-resident Lin-CD34+ cells. Importantly, a comparable level of editing was obtained upon single VLP dosing in long-term humanized mice despite them carrying higher numbers of human BM HSPC. We then explored the use of HSPC-targeted viral fusion proteins (fusogens). I will show that a CD133-targeted LV, combines potency on par with BaEVTR-LV with high specificity for CD133+ cells and that a CD117-targeted LV can transduce, in vivo, a BM cell population composing as low as 0.3% of total huCD45+ cells with a 178-fold increased specificity as compared to broad fusogens. These results set the basis for generating a potent candidate for in vivo delivery of genetic payloads to HSPC with high efficiency and specificity.

Bio:

Dr. Marianna Prokopi-Demetriades is a biotech innovator and entrepreneur, recognized for her pioneering contributions to improving oncology patient care. Marianna has recently graced the stage as a TEDx speaker, sharing her insights and spreading ideas that encapsulate her vision for the future of biotech and personalized medicine. She is the cofounder of three biotech enterprises, including RSL Revolutionary Labs Ltd, leading the creation of dermaceuticals that alleviate the side effects of cancer treatments. Her involvement with Promed Bioscience Ltd is transforming the bioprinting of artificial organs with 3D-atelocollagen, while Theramir Ltd is at the vanguard of precision cancer therapy utilizing nanotechnology and microRNAs.Honored with awards like Forbes 20WomenInTech Cyprus 2023 and the TechIsland Award Rising Star 2023, she’s also been recognized by the Republic of Cyprus with the Young Researcher Award in Life Sciences 2021, and Madame Figaro's Woman of the Year in Innovation 2020. Dr. Prokopi-Demetriades’s impact is felt well beyond the research lab. She is an esteemed CIM Honorary Fellow at the Cyprus Institute of Marketing  and a Clin.Assoc.Professor in Research Oncology  at the European University of Cyprus, School of Medicine. Her dedication to mentorship is evident in her participation on numerous boards and in mentoring roles with entities like the ENAVSMA Foundation, ICC Women Network Cyprus, ARIS/Deloitte, JA Junior Achievement Cyprus and Europe, The STEAM Academy, Falling Walls Foundation Female Science Talents, and the King’s Leadership Mentoring Programme. Her strategic vision has been instrumental in securing investment for a cutting-edge biotech hub in Limassol, demonstrating her unwavering commitment to innovation and the global advancement of the biotech industry.

Title:

Bridging Boundaries: The Pursuit of Innovation and Equity in Science and Entrepreneurship

Abstract:

Dr. Marianna Prokopi-Demetriades draws from her dynamic journey spanning clinical research in oncology and her role as a biotech entrepreneur to deliver insights into the science-industry nexus. She recounts the founding of three biotech ventures, marking her shift from academic research to the entrepreneurial world—a move characterized by embracing new challenges and showcasing strategic vision. Operating from Cyprus, she outlines the distinctive dynamics of driving innovation on a small island enriched by ICT, providing a balanced perspective on its unique advantages and limitations.

Her dedication to advancing women in science and entrepreneurship shines through as she highlights her involvement with empowering initiatives like Falling Walls Female Science Talents and ICC Women Network Cyprus. Dr. Prokopi-Demetriades addresses gender disparities in STEM fields, sharing personal encounters with bias and the proactive measures necessary for fostering inclusivity. The talk aims to be a wellspring of guidance for researchers at various career phases, advocating for persistence and the ability to adapt in an ever-evolving landscape. She stresses the importance of sustaining professional drive while nurturing personal well-being, suggesting strategies to maintain this equilibrium. Participants are invited to delve into Dr. Prokopi-Demetriades's experiences, extracting valuable takeaways for surmounting obstacles and seizing opportunities that arise at the intersection of science and business.

Senior Field Application Scientist, Cell & Gene Therapy Specialist, Europe & Middle East

Bio:

Marianna is a Senior Field Application Scientist and Cell Therapy Specialist for Europe at MaxCyte, where she supports the scientific activity of customers, collaborators, and prospects. She joined the company in December 2019, after finishing her post-doc on CRISPR-Cas9 and AAV6 HDR-mediated gene correction of IL7RA-deficient hematopoietic stem cells at the UCL Great Ormond Street Institute of Child Health. Her Ph.D. work was focused on the pre-clinical development of HIV-resistant genome-edited T cells using TALEN nucleases at the Albert Ludwig University of Freiburg. Marianna holds two patents on genome and epigenome editing of clinically relevant cells and several publications in high-impact factor journals such as Nature Biotechnology, Nucleic Acids Research, and Nature Communication.

Title:

High Efficiency Cell Engineering with the MaxCyte Electroporation Platform from Concept to Clinic

Abstract:

Genome engineering of HSPCs and T cells holds great promise for the treatment of diseases including cancer or genetic disorders. However, viral T cell engineering has several limitations, including higher cost, lengthy manufacturing process, and the risk associated with viral integration into the host genome.
Therefore, a non-viral genome editing approach is gaining attraction from basic to clinical research. MaxCyte’s clinical-scale flow electroporation efficiently delivers genome editing tools in the form of DNA, mRNA, and RNP into a wide variety of clinically relevant cells. Here, we demonstrate high editing efficiency using Maxcyte electroporation to deliver different gene editing platforms into HSPCs, resting and activated T cells for knock-out or knock-in applications maintaining high cell viability.

Associate Scientist

Bio: Dr. Petros Patsali, BSc, MSc, PhD, Associate Scientist: Petros Patsali obtained his BSc degree in Biology (Biomolecular Sciences and Biotechnology Direction) from University of Crete, Greece (2010), his MSc degree in Molecular Medicine from Imperial College London, UK (2011) and his PhD in Molecular Biology and Gene Therapy from King’s College London, UK (2017). He has the position of the Associate Scientist at the Department of Molecular Genetics Thalassemia. His doctoral and post-doctoral research focused on the development of innovative gene therapy approaches for β-thalassemia, leading to groundbreaking publications in reputable scientific journals that demonstrated expertise in the field. In addition to research contributions, Petros Patsali has been actively involved in teaching and mentoring. As a lecturer at the CING postgraduate school, he has imparted knowledge and supervised the work of post-graduate students. Petros Patsali was an elected board membership in the Cyprus Society of Human Genetics from 2020-2023. He has authored numerous international oral and poster presentations, as well as 12 additional original articles and 1 review article, with 5 articles as the first author and one as equal last author, in renowned scientific journals.

Title:

Preclinical validation of HBBIVSI-110(G>A)-specific gene editing as advanced therapy for thalassaemia

Abstract:

Patsali, P 1, Constantinou CG 1, Paschoudi, K2,5, Papaioannou N 1, Naiisseh B 1, Papasavva, PL 1, Christofi, P 2,5, Christou, S 6, Sitarou, M 6, Pirovolaki, A 6, Hadjigabriel, M 6, Athanasopoulos T 7, Mussolino C 3,4, Cathomen, T 3,4, Kleanthous, M1, Psatha, N 2, Yannaki, E 5, Lederer, CW1

1 Department of Molecular Genetics Thalassamia, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
2 School of Biology, Department of Genetics, Development and Molecular Biology, Aristotle University of Thessaloniki, Thessaloniki, Greece,
3 Center for Chronic Immunodeficiency, Medical Center – University of Freiburg, Freiburg, Germany,
4 Institute for Transfusion Medicine and Gene Therapy, Medical Center – University of Freiburg, Freiburg, Germany,
5 Gene and Cell Therapy Center/Hematology-Hematopoietic Cell Transplantation Unit, G. Papanikolaou Hospital, Thessaloniki, Greece,
6 Thalassaemia Centre, Cyprus Ministry of Health, Cyprus,
7 Gene & Cell Ltd, London, United Kingdom

E-mail: [corresponding author: [email protected] and presenting author: [email protected]]
Introduction
β-Thalassemia is a common single-gene disorder worldwide caused by deficient production of β-globin. One of the most prevalent β-thalassemia mutations is HBBIVSI-110(G>A), which creates an aberrant intronic splice site in β-globin. This mutation has a relative carrier frequency of 76% in Cyprus and above 20% in many EU countries. To address this mutation, a proof of concept for an efficient mutation-specific therapy was established using designer nucleases, including CRISPR/Cas9 RNA-guided nuclease (RGN) and TALENs. Specific DNA cleavage of the intronic mutation allows the prevailing non-homologous end joining mechanism to destroy the aberrant splice site and restore normal splicing and HBB expression. This approach achieved clinically relevant efficiencie

National Institute of Chemistry, Slovenia

Professor in Molecular Genetics, UCL Great Ormond Street Institute of Child Health

Stephen Hart obtained his PhD in Microbiology from the University of Cape Town in 1991 then undertook a postdoc at St. Mary’s Biochemistry and Molecular Genetics Department before joining the UCL Great Ormond Street Institute of Child Health (ICH) as a postdoc then Lecturer. He was promoted to professor in 2012 and has been Deputy Head of the Genetics and Genomic Medicines Department at ICH. He has worked in the field of genetic therapies for more than 20 years with a particular focus on non-viral therapies and has more than 100 publications to his name. His current research activities include the development of RNA and gene editing therapies, for the treatment of cystic fibrosis, primary ciliary dyskinesias and neuroblastoma. His group have developed novel synthetic nanoparticles for the delivery of nucleic acid therapeutics including siRNA, messenger RNA and CRISPR/Cas9 formulations. He is the lead investigator on a Strategic Research Centre grant funded by the Cystic Fibrosis Trust and Cystic Fibrosis Foundation, developing CRISPR gene editing therapies for cystic fibrosis. In 2017 he was elected to the board of directors of the American Society of Gene and Cell Therapy, serving until 2021. He is a Senior Editor for the journal Annals of Human Genetics, a member of the editorial board for the journals Gene Therapy and Genes. He is the named inventor on nine patents concerning nanoparticle delivery formulations and was the scientific founder of Nanogenics Ltd, a UCL spin-out company, commercialising nanoparticle delivery formulations for genetic therapies.

Presentation Abstract

CRISPR/Cas9 Deletion of a Deep-Intronic Splicing Mutation Followed by NHEJ Repair in CFTR as a Potential Therapy for Cystic Fibrosis

Cystic Fibrosis is an autosomal recessive disorder caused by mutations in the CFTR gene. The 10th most common CFTR mutation, 3849+10kb C>T, generates a cryptic splice site within an intron, introducing a pseudoexon leading to a truncated CFTR protein due to an in-frame nonsense mutation in the pseudoexon. We explored a targeted excision strategy to delete the pseudoexon by CRISPR/Cas9 with dual guide RNAs targeting PAM sites flanking the mutation site, followed by non-homologous end joining (NHEJ) repair. NHEJ is a more efficient pathway than HDR, and allows editing of post-mitotic cells, such as the surface epithelial cells of the airways that are involved in CF. Indels introduced by inaccurate NHEJ repair, being intronic, should have no effect on CFTR expression. We developed a non-viral, receptor-targeted nanocomplex (RTN) formulation comprising a mixture of epithelial targeting peptides with lipids that promote endosomal escape, to deliver pairs of gRNAs complexed with Cas9 in a ribonucleoprotein (RNP) complex. CF basal cells with the splice site mutation were transfected with RNPs. DNA Sanger sequence analysis revealed a deletion efficiency of more than 60%. while electropherogram analysis of mRNA revealed that canonical CFTR mRNA splicing was restored, leading to normal length CFTR protein production. Edited CF cells in air-liquid interface (ALI) cultures were analysed in Ussing chamber for functional restoration of CFTR-mediate chloride ion transport indicating partial correction sufficient for therapeutic efficacy. We evaluated in vivo lung epithelial editing of RNPs with our nano formulation in the Ai9 mouse model where CRISPR/Cas9 targeted deletion of an upstream stop codon cassette was shown to restore expression of tdTomato in the airway surface epithelium. This approach could be used to correct several other deep-intronic mutations for CF as well as >75 other genetic disorders.

Synthego, CA, USA

Aarhus University, Denmark