Clinical Applications of Genome Editing Technology PDF

Clinical Applications of Genome Editing Technology Dr. Zahi Damuni Professor of Biochemistry [email protected] PointSolutions ID: damuni Reading: Lippincott Reviews in Biochemistry, 8th Ed., chapter 34 1 1 Learning Objectives 1. Distinguish between enzyme replacement therapy, gene therapy and...

Clinical Applications of Genome Editing Technology Dr. Zahi Damuni Professor of Biochemistry [email protected] PointSolutions ID: damuni Reading: Lippincott Reviews in Biochemistry, 8th Ed., chapter 34 1 1 Learning Objectives 1. Distinguish between enzyme replacement therapy, gene therapy and gene editing. 2. Identify the critical components of the CRISPR/Cas9 system and explain why it is often the preferred method for genome editing. 3. Illustrate the application of genome editing in the targeted treatment of human diseases including Sickle Cell Anemia, Hemophilia A, Hemophilia B, Transthyretin Amyloidosis, and other diseases. 4. Describe experimental applications of genome editing in animal experimental models. 5. Distinguish between gene knock-in and knock-out and illustrate the application of such methods. 2 2 Guiding Questions What is Enzyme Replacement Therapy (ERT)? How does ERT differ from Gene Therapy and Gene Editing? What are the advantages and disadvantages of using viral vectors to deliver genes? What cellular process is required for efficient gene therapy? Which gene editing system is most used today and why? Can you compare the use of ERT, gene therapy and gene editing in clinical medicine today? 3 3 Therapies Enzyme Replacement Therapy A. Clone the missing or defective protein • Produce the enzyme or factor in vitro tissue culture • Purify the enzyme or factor • Infuse into patient routinely (weekly to monthly) ↳ target Gene Therapy A. Infect patient with a virus modified to contain the missing or defective gene • Virus’ gene cloned with incorporated gene • During viral infection, viral genome inserted into patient’s genome B. Isolate patient or donor stem cells and use them to replace defective cells • Alter genome in patient’s stem cells, typically embryonic stem cells • Implant altered cells in patient to grow throughout rest of their life 4 4 A. Enzyme Replacement Therapy • Infuse into patient routinely (weekly to monthly) • Clotting factor deficiency • Factor VIII deficiency (Hemophilia A) • Factor IX deficiency (Hemophilia B) Targeting the missing protein to the • Factor XI deficiency (Hemophilia C) correct cellular location (organelle) is • Exocrine pancreatic Insufficiency often difficult since most endocytosis • Glycogen Storage disease leads to lysosome breakdown of the • Pompe (-1,4-glucosidase) proteins. • Lysosomal storage diseases • Fabry (-galactosidase A) • Gaucher (glucocerebrosidase) • Mucopolysaccharidosis (MPS I, MPS II, MPS IVa or MPS VI) • Adenosine deaminase deficiency • 1-antitrypsin deficiency 5 Gaucher Glucocerebrosidase Fabry alpha-galactosidase A Hunter Disease 5 6 The mucopolysaccharidoses are a group of inherited metabolic diseases caused by the absence or malfunctioning of certain enzymes the body needs to break down molecules called glycosaminoglycans—long chains of sugars (carbohydrates) in each of our cells. • • • for iduronate-2-sulfatase changes a single amino acid Arg468Gln. The mutated enzyme is inactive and heparan - and dermatan sulfates cannot be broken down. Causes severe mucopolysaccharidosis – previously fatal, but a recombinant enzyme “Elaprase” is now available 6 Gene Therapy • Goals • Get a functioning copy of a defective gene into cells of a patient with a genetic disease (sickle cell, muscular dystrophy, etc.) • Get intact tumor suppressor genes into cancer cells • Get anti-inflammatory genes into joints of arthritis patients • Destroy DNA of an inserted retrovirus (e.g., AIDS) • Delivery of a gene into a cell for therapy • • • Viral vector integration Gene editing In most cases, these treatments are applied to cells from the patient in vitro that then get re-inserted into the patient. 7 7 Gene Therapies Using Virus Vectors • • How they work • Virus may integrate into genome randomly (not into the gene of interest or region) • Virus may only transiently infect cells and must be administered routinely FDA approved examples • Adenovirus-associated virus vectors • Retinal dystrophy (first approved, 2017) • HSV-1 viral vector • Recurrent melanoma with nodular lesions • Lentivirus vector • Acute Lymphoblastic Leukemia or Diffuse Large B-cell Lymphoma • Other virus or vectors • Large B-cell Lymphoma or Diffuse Large B-cell Lymphoma 8 8 Gene Therapy Challenges • • How to get foreign DNA into the cell • Liposomes: not very effective • DNA viruses or retrovirus DNA integrated into the genome • DNA viral vector or retroviral vector • Cytotoxicity due to copy number or integration locations • Designer DNA endonuclease used to direct site of integration • CRISPR/Cas9 • Older methods caused insertion in random sites of the genome, with bad side effects (e.g., cancer) • Limited to only a few cells 9 A retrovirus is a type of virus that inserts a DNA copy of its RNA genome into the DNA of a host cell that it invades, thus changing the genome of that cell. 9 Recent Advances in the Treatment of Hemophilia A and B  Haemophilia A trial results 'mind-blowing' - BBC News  AAV5–Factor VIII Gene Transfer in Severe Hemophilia A | NEJM  Transformational therapy cures haemophilia B - BBC News  Phase 1–2 Trial of AAVS3 Gene Therapy in Patients with Hemophilia B | NEJM 10 FLT180a (verbrinacogene setparvovec) is a liver-directed adeno-associated virus (AAV) gene therapy that uses a synthetic capsid and a gain-of-function protein to normalize factor IX levels in patients with hemophilia B. 10 Sickle Cell Gene Therapy Helen Obando is the youngest person (16) to get Sickle Cell Gene therapy https://www.nytimes.com/2020/01/10/theweekly/sickle-cell-dna-reset.html (Published Jan. 10, 2020, Updated Feb. 17, 2020) 11 HIV virus used 11 Summary of Therapies Enzyme Replacement Stem Cell Replacement Gene Therapy 12 12 Genome Editing Genome editing is a set of techniques aimed at changing the genome of a cell, presently only used in research and clinical trials. Uses for Biomedical Research ― Knock-out – gene disrupted or deleted ― Conditional Knock-out – spatial and temporal ― Knock-in or transgenic – gene modified or inserted ― Conditional Knock-in – spatial and temporal Uses in Medicine ― Gene therapy, both for genetic and non-genetic diseases ― Germline gene modifications 13 The DNA in germ cells (egg and sperm cells that join to form an embryo). Germline DNA is the source of DNA for all other cells in the body. Also called constitutional DNA. 13 Gene Therapy Requires Endonucleases to Cut the Genome • Zinc finger nucleases, (ZFN): DNA endonuclease fused to sequence of zinc fingers (the first technique developed, rarely used today) • TALENs: DNA endonuclease fused to a sequence of different targeting modules (An improvement on ZFN, rarely used now) • CRISPR-Cas9: Cas9 DNA endonuclease bound to a small guide RNA (sgRNA) that basepairs with the DNA and cuts host DNA (This is the method of choice today) **CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats **Cas9 = CRISPR-associated protein 9 (DNA endonuclease) 14 A zinc finger is a small protein structural motif that is characterized by the coordination of one or more zinc ions (Zn2+) in order to stabilize the fold. Zinc finger proteins are among the most abundant proteins in eukaryotic genomes. Their functions are extraordinarily diverse and include DNA recognition, RNA packaging, transcriptional activation, regulation of apoptosis, protein folding and assembly, and lipid binding. TALENs are chimeric proteins that contain two functional domains: a DNA-recognition transcription activator-like effector (TALE) and a nuclease domain. They work for gene editing by recognizing a specific sequence, which the user can design, and introducing a double-stranded break with an overhang. 14 How to Edit the Genome 1. Inject DNA into cell with a DNA endonuclease 1. DNA endonuclease cuts genome Non-homologous end joining Homologous repair a. CRISPR/Cas9 used today 2. Cell’s own DNA repair systems repair the cut using DNA added a. Non-homologous end-joining: imprecise by using DNA repair. b. Homologous repair: requires template DNA for repair 15 Non-homologous end joining is a pathway that repairs double-strand breaks in DNA. Nonhomologous end joining is referred to as "non-homologous" because the break ends are directly ligated without the need for a homologous template, in contrast to homology directed repair, which requires a homologous sequence to guide repair. 15 CRISPR/Cas9 Method 1. 2. 3. 4. Crisper Guide RNA a. Target sequence for gene of interest is cloned and expressed with a small guide RNA (sgRNA). b. Cas9 complexed with sgRNA CRISPR/Cas9 introduced into host/patient cell Cas9 makes a double stranded cut in DNA at sites that match ends of Cargo DNA. Note the requirement for protospacer adjacent motif (PAM) for Cas9. Cellular repair systems repair the DNA a. Deleting bases to render gene silent (NHEJ) b. Inserting the donor plasmid replacing original version of gene (HR or NHEJ) Left end Cargo DNA Right end Donor Plasmid + CRISPR – Cas9 1 2 2 3a 3b https://www.quora.com/How-does-CRISPR-Cas9-work Uses host cell recombination systems 16 Micro homologous end joining – The protospacer adjacent motif (or PAM for short) is a short DNA sequence (usually 2-6 base pairs in length) that follows the DNA region targeted for cleavage by the CRISPR system, such as CRISPR-Cas9. The PAM is required for a Cas nuclease to cut and is generally found 3-4 nucleotides downstream from the cut site. 16 What is PAM? • The protospacer adjacent motif (PAM) is a short DNA sequence (usually 2-6 base pairs in length) that follows the DNA region targeted for cleavage by the CRISPR system, such as CRISPR/Cas9. The PAM is required for a Cas endonuclease to cut and is generally found 3-4 nucleotides downstream from the cut site. What does sgRNA stand for and why? • Short guide RNA, because it is only up to 100 bp 17 17 CRISPR Injected into Blood Treats a Genetic Disease for the First Time Ever • Transthyretin amyloidosis is a slowly progressing condition caused by a mutation - a genetic defect in the TTR gene. • New Treatment: CRISPR injected into the blood treats a genetic disease for first time | Science | AAAS (sciencemag.org) • Landmark CRISPR trial shows promise against deadly disease (nature.com) 18 18 CRISPR/Cas9 • Can CRISPR cure Sickle-cell Disease? – YouTube https://www.youtube.com/watch?v=mQ8Ola_C5po Gene therapy in sickle cell works by knocking down the expression of the BCL11A gene to flip the switch back fully to fetal hemoglobin, simultaneously increasing fetal hemoglobin, which does not sickle, and directly reducing sickling hemoglobin. • Genome Editing with CRISPR-Cas9 • Jennifer Doudna (UC Berkeley / HHMI): Genome Engineering with CRISPR-Cas9 • Nobel Lecture: Jennifer Doudna, Nobel Prize in Chemistry 2020 • CRISPR-associated Transposase System: New Capabilities in Gene-editing • How CRISPR-Cas9 works 19 Sickle-cell anemia results from an A to T transversion in the second nucleotide of codon 6 of the beta-globin gene. Sickle cell disease is caused by a mutation in the hemoglobin-Beta gene found on chromosome 11. Jennifer Doudna and Emmanuelle Charpentier have been awarded the ultimate science prize for their breakthrough research on CRISPR technology. Learn about their scientific journey as well as the vital contributions of other scientists that paved the way for the emergence of CRISPR as the most powerful gene-editing tool. 19 CRISPR/CAS9: Transforming T-cells into a form that can hunt cancer • Non-viral precision T cell receptor replacement for personalized cell therapy | Nature • 'Leap forward' in tailored cancer medicine - BBC News 20 20 Newest way with the CAST transposase Eliminates need for host recombination 21 21 Genetic Modifications • Knock out: A gene is disrupted (artificial loss-of-function mutation). Method of choice to determine physiological function of a gene. • Knock in: A gene is changed into a different version, or a foreign gene is introduced (transgenics). Can be used to create gain-offunction mutations. • Knock down: The genome is left unchanged, but a siRNA is introduced that prevents translation of selected mRNAs. Usually transient. 22 22 Examples of Genes Edited In Mice & Rats • Cystic Fibrosis demonstrated that the Δ-F508 mutation causes cystic fibrosis in mice • Amyotropic Lateral Sclerosis (ALS) - This is a deadly disease that destroys motor neurons in the spinal cord. • Mutation G93A in the gene for superoxide dismutase-1 (SOD1) causes ALS in humans, most likely because the mutated protein forms aggregates • Knock-out of SOD1 in mouse does not mimic ALS • Knock-in of G93A in the mouse does produce an ALS model 23 23 FDA Approved Gene Therapies • All FDA approved gene therapies use stem cell to date • Use patients own stem cells • Can screen out “bad” integrations so only “good” stem cells used • Injected into site of disease • Retinal dystrophy • Melanoma nodules • General circulation • Hematopoietic gene therapies • All CRISPR-CAS9 gene therapies still in clinical trials • Block HIV infection • Sickle cell • Cancer 24 24