CRISPR PDF

20/02/2024 Please complete the National Student Survey (NSS)! thestudentsurvey.com 1 XY3121 Molecular Medicine CRISPR 21.02.24 SGM104 Dr Harry Potter [email protected] 2...

20/02/2024 Please complete the National Student Survey (NSS)! thestudentsurvey.com 1 XY3121 Molecular Medicine CRISPR 21.02.24 SGM104 Dr Harry Potter [email protected] 2 1  20/02/2024 01 Learning outcomes 1. Understand the biological origins of genome editing and CRISPR/Cas9 technology. 2. Describe the main mechanisms of CRISPR/Cas9 function. 3. Integrate the importance of CRISPR/Cas9 in basic and translational medical research. 4. Describe ongoing developments in CRISPR/Cas9 research including clinical trials and DNA methylation editing. 3 02 Epigenetics vs genetic changes Epigenetic modifications Genetic modifications Does not change DNA sequence Editing the DNA sequence 4 2  20/02/2024 03 Base pair changes (1) - eukaryotes Eukaryotic organisms – ~109 DNA bases. - How and when are these changed? Meiosis Small-scale Large-scale DNA damage mutations mutations and repair (e.g. SNPs) (e.g. CNVs) (e.g. NHEJ) 5 03 Base pair changes (2) - prokaryotes Prokaryotic organisms – susceptible to attack by e.g. bacteriophages. - How and when are base pairs changed? - Restriction enzymes (protection). - Starting point for DNA editing technology. Q: how can we make use of existing molecular machinery to edit mammalian genomes? e.g. protection from phages by restriction enzymes in viruses 6 3  20/02/2024 03 Base pair changes (3) – why do we care? Gene x environment (GxE) interactions. - Risk vs resilience for chronic disease. - Sickle cell disease, neurodevelopmental, cardiometabolic, blindness, cancer, neurodegenerative, neuropsychiatric… A lot of diseases have a large genetic component: - Huntington’s, blood cancers, breast cancer, Figure from H. Potter PhD thesis muscular dystrophy, congenital blindness… The ability to modify the DNA sequence could be a huge resource for novel therapies. 7 04 CRISPR introduction 8 4  20/02/2024 05 Limitations with mammalian genome editing Early studies on the yeast genome provided foundations for mammalian studies: - Showed that mammalian cells can incorporate DNA into their genome. - Homologous recombination. - Allowed study of genes (i.e. ‘what does this gene do if I add it to a cell?’). Several limitations to the field: Low rate of Integration rate is spontaneous dependent on Off-target effects integration cell characteristics (integration at random genomic loci) (1 in 103-109 cells) (type, state) 9 2 minute break 10 5  20/02/2024 06 Meganucleases Double-stranded breaks (DSB) increase frequency of integration. - Several orders of magnitude. Meganucleases (recognises and cut 14-40 bp stretches) - e.g. I-SceI (cuts at 18 bp). Limitations? - ~100s of endogenous meganucleases with specific recognition sequence. - Most DSBs are repaired by non-homologous end-joining (NHEJ). - NHEJ is error prone – off-target effects? 11 07 Zinc fingers Small protein motifs, regulated by Zn2+. - Bind DNA (3 bp) in a specific manner. - i.e. higher DNA binding specificity compared to meganucleases. Biotechnologically engineered zinc finger nucleases (ZFN): - Fuse to DNA cleavage domain of Fok1 (endonuclease). - Fok1 requires homodimerisation at cleavage domain. - Two ZFNs could be combined to increase DNA specificity: 64 zinc fingers Uniquely target any 18-21 bp sequence (43) Pick 6-7 ZF 12 6  20/02/2024 08 Transcription activator-like effector proteins TALE protein identified in Xanthomonas bacteria. - Uniquely identify a single base pair. - Further increases specificity of DNA targeting. - Chimeric fusion of Fok1 protein produces TALE nuclease (TALEN). Limitations with flexibility and efficiency genome editing, and protein cloning. Most recent step is the development of CRISPR/Cas9 technology. 13 09 Gene editing progress 14 7  20/02/2024 10 minute break Why not use the break for the National Student Survey (NSS)? thestudentsurvey.com 15 10 CRISPR (1) - components Clustered Regularly Interspaced Short Palindromic Repeats - Component of bacterial immune system. - Capable of cleaving DNA from invading pathogens (i.e. bacteriophages). CRISPR-associated protein (Cas; a nuclease). Single guide RNA (sgRNA; or just gRNA). 16 8  20/02/2024 10 CRISPR (2) – ‘original’ function Cas binds to & cuts DNA. - sgRNA directs Cas9 nuclease to its target. - Functions to cut DNA of viruses (bacteriophages, or ‘phages’). - Disables virus/viral DNA. Allows for double-stranded DNA breaks (DSB) at very specific locations in the genome. - How can this be used in mammalian cells? 17 10 CRISPR (3) – ‘original’ function Basis of adaptive/acquired immunity (bacteria, archaea). - Upon infection, Cas nuclease cleaves protospacer region. - Fragment is stored within bacterial genome (‘memory’). - Placed within palindromic spacers. First identified in E. coli. Upon reinfection: Upon reinfection, phage-specific - Cas9 can recognise & destroy virus. Cas9 nucleases are expressed. 18 9  20/02/2024 10 CRISPR (4) – mammalian editing 1993 – Dr Fransisco Mojica - Bacterial genome contained repetitive palindromic DNA repeats. - Interspaced with genetic material from phage genome. 2012 – Dr Jennifer Doudna and Dr Emmanuel Charpentier. - Repurposing CRISPR as genetic editing tool. - 2020 – Nobel Prize in Chemistry. 19 10 CRISPR (5) – sequence of events I sgRNA contains specific sequence that is complementary to target DNA: 1. sgRNA complexes with Cas9. 2. sgRNA directs Cas9 to sequence. 3. Cas9/sgRNA binds adjacent to DNA target. 4. DNA unwinds at target. Protospacer adjacent motif (PAM) sequence (2-6 bp long, 3-4 bp downstream from target). - Required for Cas9 binding (every ~50 bp). 20 10  20/02/2024 10 CRISPR (5) – sequence of events II 21 10 CRISPR (5) – sequence of events III Cas9 cleaves target DNA to form DSBs. - How are DSBs resolved? - NHEJ – error prone, causing addition/deletion of base pairs. Consequence? - Mutations in promoter, protein-coding sequence. Depending on how the DSBs are repaired, CRISPR/Cas9 can also be used to: - Delete DNA loci. - Correct DNA loci. 22 11  20/02/2024 10 CRISPR (5) – sequence of events IV Two DSBs (i.e. two targets). Two DSBs (i.e. two targets). Naked ends are complementary. Naked ends are complementary. Middle is removed, ends are re-joined. Middle is removed. Homology-directed repair. DNA template added – ends match - i.e. not NHEJ. the DSBs. 23 2 minute break 24 12  20/02/2024 11 Translational importance (1) Technology is readily available for use in in vitro and in vivo models. - i.e. cells, mice, rats. - Provide fundamental information on biology, safety, and efficacy. - Limited translational capacity. Ex vivo and clinical trials? 25 11 Translational importance (2) – ex vivo Cells are removed from the patient. - e.g. HSCs, T cells. CRISPR toolkit (Cas9, gRNA) is packaged as an AAV vector. - Or delivered to cells by electroporation/nucleofection or microinjection. - High-voltage shock can damage cells. - Viral toxicity, high expression leading to off-target effects. 26 13  20/02/2024 12 Clinical trial – SCD (1) Sickle cell disease (SCD) and beta thalassemia – Hb gene mutations. - SCD – sickle shaped erythrocytes; sudden-onset severe pain, anaemia. - Beta thalassemia – not enough Hb produced; fatigue, anaemia. HbF is not affected by SCD mutation. - Ex vivo CRISPR strategy (β-globin gene). - Harvest patient-derived cells. - ‘Turn on’ HbF gene. - i.v. administration of cells. 27 12 Clinical trial – SCD (2) 28 14  20/02/2024 13 Clinical trial – LCA Leber congenital amaurosis (LCA). - Most common congenital childhood-onset blindness. - Blindness within first few months postnatally. - SNP in CEP290 gene (truncated CEP290 protein). - Impaired function of photoreceptors. First in vivo CRISPR trial (2020). - Small organ – single dose, intraocular injection. - Lower risk of treatment reaching other tissues (off- target effects?) - In vivo delivery means potential for immune reactivity. 29 14 Clinical trial – CAR-T cell therapy (1) Chimeric antigen receptor (CAR) transduced into human T cells. - Genetically engineered fusion proteins. - Extracellular – single chain antibody fragment (antigen-binding). - Intracellular – T cell signal transduction. Recognition of tumour cells. - Stimulation of CAR-T cells. Combines: - Specificity of antibodies. - Toxicity of T cells. 30 15  20/02/2024 14 Clinical trial – CAR-T cell therapy (2) CD19 CAR recognises CD19 on B cells. - Clinical trial for relapsed or refractory lymphoma. - Up to 89% tumour remission across 28 month follow up period. - Effective in treating B-cell lymphoblastic leukaemia. Less reliable in treating solid tumours or other haematological cancers. PD1 (cell surface receptor) regulates T/B cell function. - Inhibition of PD1 combined with CAR-T (e.g. CD19, CD8) cell therapy shows more promising results. 31 15 Clinical trial – iPSCs in T2DM Induced pluripotent stem cells (iPSCs). - Relevance for therapeutics in many disease states (‘pluripotent’). - Neurodegenerative disease transplantation, disease modelling, drug discovery, wound healing, muscular dystrophy, haemophilia, type II diabetes… In type II diabetes mellitus (T2DM), iPSC grafts of hypoimmunogenic grafts (but still poor graft survival). CRISPR can be used for: - Disruption of: B2M, class II transactivator (CIITA; both HLA components). - Overexpression of: CD47 (dampens host phagocytic activity). 32 16  20/02/2024 16 Clinical trial – hereditary transthyretin amyloidosis (hATTR) (1) Progressive, fatal, multi-organ system disease. - Acquired or hereditary (autosomal dominant). - Any of >100 mutations on TTR gene. - Dissociation of TTR tetramers into insoluble monomers. - Protein misfolding leads to toxic amyloid fibril aggregates in extracellular tissue (neural, myocardial). - Neuropathy, cardiomyopathy. Liver transplants (with w/t TTR) is available but limited: - Donor availability. - Lifelong immunosuppressive drugs. 33 16 Clinical trial – hereditary transthyretin amyloidosis (hATTR) (2) Several ongoing CRISPR clinical trials. Knock out TTR gene in hepatocytes. - Phase I clinical trial. - 6 patients with polyneuropathy. - 28 days post-treatment, up to 87% reduction in circulating TTR levels in high dose group. 34 17  20/02/2024 17 Methylation & CRISPR Q: can we use CRISPR/Cas9 technology to manipulate epigenetic modifications? - E.g. fuse deactivated Cas9 (dCas9) with catalytic domain of DNA methyltransferase 3A (DNMT3A). - Single guide RNA (sgRNA) allows for gene- specific methylation. Potential to control gene expression. - Off-target effects? - Other consequences of altering DNA methylation? 35 18 Interactive guide 36 18  20/02/2024 19 Examples of CRISPR clinical trial publications Primary papers (browse for wider reading): [SCD] Frangoul et al. (2021). CRISPR-Cas9 gene editing for sickle cell disease and beta-thalassemia. N Engl J Med. 384:252-260. [LCA] Ruan et al. (2017). CRISPR/Cas9-mediated genome editing as a therapeutic approach for Leber Congenital Amaurosis 10. Mol Ther. 25:331-341. [PD1/CAR-T] John et al. (2013). Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells. Clin Cancer Res. 19:5636-5646. [iPSCs in T2DM] Deuse et al. (2019). Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients. Nat Biotechnol. 37:252-258. [hATTR] Gillmore et al. (2021). CRISPR-Cas9 in vivo gene editing for transthyretin amyloidosis. N Engl J Med. 385:493-502. Reviewed in (read more thoroughly): Zhang et al. 2023. Current trends of clinical trials involving CRISPR/Cas systems. Front Med. 10:1292452. 37 Activity – CRISPR ethical considerations (15 mins) Pre-clinical Clinical Animal or cell models Translatability, patient safety, outcomes Risks Benefits Challenges 38 19  20/02/2024 Please complete the National Student Survey (NSS)! thestudentsurvey.com 39 20