MBBS1 Genomics 2024 Antoniou PDF

Module: 6BBYG305 – Medical Genetics Gene Therapy Clinical Trials Employing Viral Vectors and Gene Editing Approaches for Inherited Single Gene Disorders -A Translational Introductory Overview- Prof Michael Antoniou Department of Medical and Molecular Genetics Gene T...

Module: 6BBYG305 – Medical Genetics Gene Therapy Clinical Trials Employing Viral Vectors and Gene Editing Approaches for Inherited Single Gene Disorders -A Translational Introductory Overview- Prof Michael Antoniou Department of Medical and Molecular Genetics Gene Therapy Clinical Trials Employing Viral Vectors and Gene Editing Approaches for Inherited Single Gene Disorders -A Translational Introductory Overview- Aims and objectives § To understand how viral vector gene therapy can be delivered both in vivo and ex vivo § To understand the principles behind vector selection and design for a given disease condition § Appreciate initial outcomes from in ex vivo and in vivo clinical trial applications of CRSPR/Cas gene editing § To appreciate the large breadth of conditions currently being targeted by gene therapy in clinical trials § To be able to critically evaluate of the outcomes of clinical trials § Wirth T, Parker N, Ylä-Herttuala S (2023) History of gene therapy. Gene, 525: 162-169. doi: 10.1016/j.gene.2013.03.137  What is gene therapy? “The most comprehensive description of gene therapy is therefore the use of recombinant genetic material (DNA, RNA or hybrid molecules) under different forms or pharmaceutical preparations, as a therapeutic agent.” European Society of Gene and Cell Therapy, position paper on gene therapy, 2002. Somatic Gene Germ Line Gene Therapy Transfer Three studies found unwanted changes surrounding the target gene; rearrangements, large deletions, chromosomal loss Ledford H, Nature, 583: 17-18, 2020 Human Gene Therapy Trials Worldwide : 1989-2018 http://www.wiley.com/legacy/wileychi/genmed/clinical/ First gene therapy success in clinical trials in the year 2000 Up to year 2000 all gene therapy clinical trials were failures Human Gene Therapy Trials Worldwide : 1989-2023 J Gene Med; https://a873679.fmphost.com/fmi/webd/GTCT British Society for Gene & Cell Therapy: https://www.bsgct.org/ European Society for Gene & Cell Therapy: https://www.esgct.eu/ American Society for Gene & Cell Therapy: https://www.asgct.org/  Approved Gene Therapy Clinical Trials Ginn SL et al. (2018) Gene therapy clinical trials worldwide to 2017: An update. J Gene Med. 20: e3015 Modes of delivery and cell/tissue targets Gene Therapy Delivery Protocols in vivo: systemic delivery or direct injection into affected organ ex vivo: isolate cells from patient and place in tissue culture, genetically correct cells with vector (usually viral vector), return cells to patient Kaufmann KB et al. (2013) EMBO Mol Med. 5: 1642-1661  Gene Therapy Procedures In Vivo Gene Delivery to the Eye  Gene Therapy Procedures Ex Vivo Gene Delivery to HSC GLP tissue culture facility Cells grown in tissue culture bags Common Components of a Gene Therapy Protocols Efficient Gene Transcriptional Target Tissue Delivery Control Accessible Viral vectors Sufficiently high Manipulable Non-viral vectors Sustained 1. Mutually interdependent components; requirements within one area puts restraints on the others. 2. Therapeutic requirements for a given disease will dictate technical details. 3. No one procedure will fulfil the needs of all diseases. 4. Three types of gene therapy outcomes: Gene silencing using siRNA/shRNA/miRNA Gene replacement/addition Gene editing mediated gene disruption / gene correction  Target Tissues Haematopoietic System Muscle Liver Vasculature Skin CNS Lung Stem cells Neurons Myoblasts Endothelium Keratinocytes Progenitor cells Hepatocytes Glia Epithelium Myofibres Smooth muscle Fibroblasts Mature cells Microglia Problems: - Access - Non-dividing - Ex vivo procedure only Ideal cell target: pluripotent, self-regenerating stem cells from the patient being treated.  Sources of Stem Cells Bone marrow* Epidermis (skin)* Skeletal muscle Neural (numerous areas of CNS) Adipose *In clinical trials or approved Gene Delivery: Viral Vectors Non-integrating: adenovirus, AAV, HSV Integrating: retrovirus/lentivirus; long-term expression possible Efficiency of delivery: high Problems: § Retroviral transduction; dividing cells only § Genetic size limitation § Mostly non-targetable; local delivery in vivo § Systemic delivery of some AAV vector serotypes; preferential tissue transduction § Ex vivo applications with retroviral/lentiviral vectors Immunogenicity: can be very high; e.g. adenovirus Manufacture: difficult to produce and store; expensive Retroviral/Lentiviral Vector Design HIV lentiviral genome (ssRNA) Remove HIV genes; replace with therapeutic transgene or other gene of interest Vector entry by receptor mediated endocytosis Gene Addition Gene Therapy Clinical Trials Successes via Ex Vivo Procedure § Severe combined immune deficiencies (SCID): § SCID-X1; SCID-ADA; Wiskott-Aldrich Syndrome (WAS) § X-linked chronic granulomatous disease (CGD) § Junctional epidermolysis bullosa (JEB) § X-linked adrenoleukodystrophy (ALD) § Metachromatic leukodystrophy (MLD) § b-thalassaemia/sickle cell disease (b-thal/SCD) § CAR-T cell therapy for B-lymphoma All the above been at least partially successfully treated using either a gammaretroviral (SCID, CGD, JEB, WAS, CAR T cell) or lentiviral (ALD, MLD, WAS, b-thal/SCD) vector via an ex vivo procedure targeted to haematopoietic stem cells in the case of SCID-X1/ADA, CGD, ALD, MLD, WAS and epidermal stem cells in the JEB. T cells targeted in CAR T cell therapy. ADENO-ASSOCIATED VIRUS (AAV) VECTORS AAV genome; single AAV vector: replace stranded DNA viral genes with therapeutic gene cassette Vector entry by receptor mediated endocytosis Sayed N et al. (2022) Gene Addition Gene Therapy Clinical Trials Successes via In Vivo Procedure Haemophilia B Leber’s congenital amaurosis Spinal muscular atrophy (SMA) Duchenne muscular dystrophy (partial) All the above have used an AAV vector delivered directly to desired tissue All approved for clinical use Human Gene Therapy Clinical Trials 2021 Vectors Employed § >26% use gamma-retroviral or lentiviral vectors § 8% AAV vectors Genome editing gene therapy applications  Genome Editing Gene Therapy Megan D. Hoban and Daniel E. Bauer (2016) A genome editing primer for the haematologist. Blood 127: 2525-2535 Genome editing using site-directed nucleases - SDNs  Diverse Applications of CRISPR/Case Genome Editing NHEJ repair; INDEL formation; gene knock-out Double strand DNA break HDR; gene modification or insertion; need template DNA Nuclease defective Cas9; Base editing gRNA/Cas-base editor (eg cytodine deaminase) fusion Nuclease defective Cas9; gRNA/Cas-DNA Epigenetic modifications methyltransferase (eg DNMT3A) or Cas-histone modifier (eg p300) fusions  Currently recognized genome editing unintended mutational effects Recognized gene editing off-target mutational effects: § Unintended alterations or mutations to other genes in addition to the target gene(s). Note: off-target mutations will not be random but in other genes. Recognized gene editing on-target mutational effects: Large DNA deletions and rearrangements affecting more than one gene: Kosicki M et al. Nat Biotechnol., 36: 765, 2018; Ledford H, Nature, 583: 17-18, 2020. Creation of new gene sequences resulting in new RNA and mutant proteins: Smits AH et al. Nat Methods, 16: 1087-1093, 2019; Tuladhar R et al. Nat Commun., 10: 4056, 2019. Chromothripsis: Chromosome shattering and random rejoining Leibowitz, M.L et al. (2021) Nat Genet 53: 895-905. Unintended insertion of plasmid repair template or contaminating foreign DNA: Skryabin BV et al. Sci. Adv. 6: eaax2941, 2020; Ono R et al. Commun Biol. 2: 57: 2, 2019  Genome Editing Clinical Trials Innovative Genome Institute (IGI) UC Berkeley/UCSF CRISPR/Cas mediated correction of SCD mutation FDA clinical trial approval, Mar 2021 https://news.berkeley.edu/2021/03/30/fda-approves-first-test-of-crispr-to-correct-genetic-defect-causing-sickle-cell-disease/ Sheridan C (2018). Nat Biotech. 36: 907-908 https://innovativegenomics.org/news/crispr-clinical-trials-2022/  Base editors hit the clinic Nature NEWS; 13 July 2022 https://www.nature.com/articles/d41573-022-00124-z Nature News 13 November 2023 First trial of ‘base editing’ in humans lowers cholesterol — but raises safety concerns Super-precise gene-editing approach switches off a gene in the liver that regulates ‘bad’ cholesterol. Miryam Naddaf https://www.nature.com/articles/d41586-023-03543- z?utm_medium=affiliate&utm_source=commission_junction&utm_campaign=CONR_PF018_ECOM_GL_PHSS_ALWYS_DEEPLINK&utm_c ontent=textlink&utm_term=PID1612532&CJEVENT=ad837e6386e511ee82dc78b20a18b8fb&countryCode=de CRISPR/Cas/Base editing trial targeting patients with HeFH Patient group: heterozygous familial hypercholesterolemia (HeFH). Mutations in APOB, LDLR, Naddaf M Nature, LDLRAP1, PCSK9. Loss of control 623: 671, 2023 in LDL-C. Gene target: PCSK9 in the liver Agent: CRISPR/Base editor (mRNA/gRNA); lipid nanoparticle complex; systemic delivery Phase Ib trial outcomes: § Up to 84% reduction in PCSK9 § 55% reduction in LDL cholesterol after 28 days; persisted for 6 months Adverse outcomes: https://www.vervetx.com/sites/default/files/2023- 11/Verve_AHA_2023_LBS_for%20website.pdf One death after 5 weeks One death after 1 day Ex vivo gene therapy targeting haematopoietic stem cells using retroviral vectors  Gene Therapy for Primary Immunodeficiencies (PIDs) Reference: Fischer A, Hacein-Bey-Abina S (2020) Gene therapy for severe combined immunodeficiencies and beyond. J Exp Med. 217: e20190607 Gene Therapy of X-SCID Paris and London  X-SCID vX-linked Severe Combined Immunodeficiency. vComplete absence of NK and T-cells in the peripheral blood. vMutation of gamma chain (gc; IL2RG), a sub-unit of haematopoietic cytokine receptors IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. Result: block in T cell development. v1:50-100,000 births. Children with X-SCID die in the first year of life. Regular treatment: §residence in sterile chamber §antibiotics §bone marrow transplant - cure  Gene Therapy of X-SCID Gene Delivery Procedure MFG: Maloney murine leukaemia virus-based gammaretroviral vector y LTR IL2RG cDNA LTR pA Ex vivo gene delivery protocol: §BM harvested; CD34-positive stem cells isolated. §Cells activated to grow for 24 hours with cytokines. Note: need to activate mitosis as retroviruses will only transduce dividing cells. §Cells transduced by three rounds of culture in the presence of the supernatant containing the IL2RG retroviral vector in sterile bags. §Transduced CD34+ cells infused back into patient.  Gene Therapy of SCID-X1 Clinical Outcome Longitudinal study of lymphocyte subsets from patient 1 (P1) and patient 2 (P2). Absolute counts of T cells (CD3+, CD8+, and CD4+), B cells (CD19+), and NK cells (CD16+, CD56+) are shown as a function of time. Day 0 is the date of treatment. The scale for NK cells is on the right hand side of each panel. P1 - 11 months; P2 - 8 months. NOTE: selective survival advantage of gene corrected cells. Cavazzana-Calvo M et al. (2000) Science, 288: 669-672.  Gene Therapy of X-SCID Current Status of Clinical Trials – London and Paris 20 infants treated: 10 infants - France 10 infants - UK All infants responded well, progressing to normal immune responses and growth rates. The 2 oldest are now 20 years since treatment and ~23 years old.  Severe adverse events in clinical trials for SCID-X1: Insertional Mutagenesis from Onco-retroviral Vectors A total of 6 patients have developed T cell acute lymphoblastic leukaemia 2– 14 years after treatment; five in Paris, one in London; fatal in one Retroviral vector integration site 35kb upstream of LMO2 Howe SJ et al. (2008) J Clin Invest., 118:3143-3150. CLEAR NEED FOR IMPROVED VECTOR DESIGN WITH REDUCED INSERTIONAL MUTAGENESIS POTENTIAL [Use of self-inactivating (LTR U3 deleted) retroviral & lentiviral vectors…..]  Gene Therapy of SCID-ADA Genetics and Pathology SCID-ADA - deficiency of adenosine deaminase (ADA). Purine metabolic defect; accumulation of toxic metabolites adenosine/deoxy-adenosine; leads primarily to impaired lymphocyte development and function. Normal treatment: enzyme replacement therapy bone marrow HSC transplant Gene Therapy § Accumulation of toxic metabolites and death of uncorrected cells § Selective advantage to cells that produce sufficient vector-derived ADA  Gene Therapy of SCID-ADA Gene Delivery Vector y LTR ADA cDNA NeoR LTR GIADA retroviral vector pA Ex Vivo Gene Delivery Procedure vBM CD34+ cells isolated vNon-myeloablative conditioning (busulfan chemotherapy) Aiuti A et al. (2002) Science, 296: 2410-2413. Gene Therapy of SCID-ADA: Clinical Outcome Hematopoietic and lymphoid reconstitution after gene therapy. (A and B) Absolute neutrophil counts (solid diamonds) (left ordinate) and platelet counts (open circles) (right ordinate) of Pt1 and Pt2 before and after gene therapy. Bu indicates the 2 days of busulfan administration; arrows show the date of infusion of transduced CD34+ cells (day 0). (C and D) Total lymphocyte counts (solid circles) (left ordinate) and white blood cell counts (open circles) (right ordinate) in the PB. (E and F) Absolute counts of PB CD19+ B cells (open circles), CD3+ T cells (solid diamonds) (left ordinate), and CD56+/CD16+ NK cells (solid triangles) (right ordinate). (G and H) Absolute counts of PB CD3+/CD4+ T cells (solid squares), CD3+/ CD8+ T cells (solid triangles), CD4+/CD45RA+ naive T cells (open squares), and numbers of TREC (solid circles) in CD3+ cells. The scale for TREC numbers is on the right. TREC levels in age-matched controls are 270 ± 130 copies per 100 ng of DNA. Aiuti A et al. (2002) Science, 296: 2410-2413. Strimvelis: second approved gene medicine in EU (2016) (http://www.ema.europa.eu/ema/index.jsp?curl=pages/medic ines/human/medicines/003854/human_ med_001985.jsp&mid=WC0b01ac058001d124; http://www.bionews.org.uk/page_656359.asp) § Retroviral vector encoding adenosine deaminase (ADA) § Targets patients with ADA-SCID § Ex vivo bone marrow stem cell correction NOTE: this procedure does NOT address the systemic toxicity from adenosine & deoxyadenosine and damage to the CNS. Ex vivo gene therapy targeting epidermal (skin) stem cells using retroviral vectors  Gene therapy for Junctional epidermolysis bullosa (JEB) Mutations in genes encoding the basement membrane component laminin 5 (LAM5), a devastating and often fatal skin adhesion disorder; LAM5-beta3-deficiency treated Maviolio F et al. (2006) Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells. Nat Med. 12: 1397-1402. Review: Abdul-Wahab A, Qasim W & McGrath JA (2014) Gene therapies for inherited skin disorders. Semin Cutan Med Surg., 33: 83-90. Gene therapy for Junctional epidermolysis bullosa (JEB) Patient: KEP25; 36-year-old male. Most of his body was covered by large, hard-to-heal blisters or infected crusts, with few moderately affected areas. Ex vivo: Epidermal stem cells: biopsy from palms Retroviral vector: y LTR LAMB3 cDNA LTR pA Transplantation: four and five genetically modified grafts, each 55 cm2 (a total of ~500 cm2), on the right and left legs, respectively Gene therapy for Junctional epidermolysis bullosa (JEB) SHOW VIDEO 3.5 cm. Graft  Gene therapy for Junctional epidermolysis bullosa (JEB) 3.5 cm. Graft Complete epidermal regeneration on both legs at day 8; normal-looking epidermis was maintained throughout the 1-year follow-up. Full, normal and robust skin function restored. Gene therapy for Junctional epidermolysis bullosa (JEB) De Rosa L et al. (2021) Front. Genet. 12: 705019. https://doi.org/10.338 9/fgene.2021.705019 Revolutionary breakthrough: FDA approves Vyjuvek, the first topical gene therapy for dystrophic epidermolysis bullosa [Khan A et al. (2023) Ann Med Surg (Lond). 85: 6298-6301] Epstein AL, Haag-Molkenteller C (2023) Cell, 186: 3523-3523.e1  Gene Therapy for the Haemoglobinopathies Thalassaemia - globin chain imbalance § a-thalassaemia - not enough a-chains § b-thalassaemia - not enough b-chains b-globin structural mutation: normal amounts of b-globin gene expression but functionally defective b-globin chain §Sickle cell disease/anaemia: glutamic acid to valine at codon 6 Current Therapies for the Haemoglobinopathies Red blood cell transfusions and iron chelation therapy. Haematopoietic Stem Cell (HSC) transplant: 30% of cases: bone marrow cord blood Gene therapy for b-thalassaemia and sickle cell disease Procedure: § Ex vivo § Bone marrow haematopoietic stem cells (HSCs) Gene addition: § Lentiviral vectors Genome editing: § Reactivation of the foetal g-globin genes (HBG) § Correction of inherited mutation Gene Therapy for the b-thalassaemia and sickle cell disease (SCD) Activation of the foetal g-globin genes to compensate for lack of b-globin in b- thalassaemia or defective b-globin in SCD  Reactivation of the foetal g-globin genes (HBG): knockdown/inhibition of BCL11A BCL11A: § Key repressor of HBG expression § Knockdown/inhibition of BCL11A – activation of HBG expression § Compensation for lack (b-thalassaemia) or faulty (SCD) b- globin (HBB) § shRNA-mediated knockdown of BCL11A expression § Genome editing knockdown of BCL11A expression § Genome editing mutation of BCL11A binding sites in g-globin gene promoters  Reactivation of the foetal g-globin genes (HBG): knockdown/inhibition of BCL11A § shRNA-mediated knockdown of BCL11A expression Esrick EB et al. (2021). New England Journal of Medicine, 384: 205–215. Six patients (colour coded lines in graphs) Median follow-up 18 months (range, 7 to 29) All engrafted; adverse events from chemotherapy. All fully evaluated patients achieved robust and stable HbF induction: Percentage HbF/(F+S) at most recent follow-up, 20.4 to 41.3% HbF broadly distributed in red blood cells (F- cells 58.9 to 93.6% of untransfused red cells) HbF per F-cell 9.0 to 18.6 pg per cell Clinical manifestations of sickle cell disease: reduced or absent during follow-up  BCL11A Knockdown Gene Therapy for Sickle Cell Disease and b-thalassaemia § Genome editing knockdown of BCL11A expression Procedure: § CRISPR-Cas9 knockdown of erythroid expression of BCL11A gene by disrupting erythroid enhancer §Ex vivo manipulation of patient’s own HSC procedure §Full chemotherapeutic myeloablation (to destroy HSC carrying SCD mutation and still residing in patient’s bone marrow Frangoul H et al. (2021) N Engl J Med. 384: 252-260  CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia Frangoul H et al. (2021) N Engl J Med. 384: 252-260 Thal-major Patient [No RBC transfusion from 30-days after gene therapy] HbF: 0.3g/dL to 13.1g/dL F-cells: 10.1% to 99.7% month 6 month 18 (blue bars). and maintained (grey bars). SCD patient [No vaso-occlusive crises during 16.6- month follow-up] Hb: 7.2g/dL to 12g/dL month F-cells: 99.9% month 5 15. HbF 9.1%/HbS 74.1% to HbF and maintained (grey Note: adverse events 43.2%/HbS 52.3% month 15 bars). from myeloablation which (blue bars). resolved with treatment.  CTX001 clinical trial outcomes: 28/29 SCD patients no longer had VOC; 39/42 b-thalassaemia patients no longer needed RBC transfusions. https://www.science.org/content/article/united- kingdom-approves-first-ever-crispr-treatment-cure- BBC News, Nov 16, 2023 sickle-cell-disease-and- beta?utm_source=sfmc&utm_medium=email&utm https://www.bbc.co.uk/news/health-67435266 _content=alert&utm_campaign=WeeklyLatestNews &et_rid=17151260&et_cid=4989931 Gene Therapy for the b-thalassaemia and sickle cell disease Gene addition therapy Globin Lentiviral Gene Therapy Vectors b-globin mini-gene Locus Control Region elements y cppt D LTR HS2 HS3 HS4 SIN-LTR pA Lentiviral Gene Therapy Vectors for the b-haemoglobinopathies Ferrari G et al. (2017) Hematol. Oncol. Clin. N Am. 31: 835-852. b-thalassaemia Gene Therapy : Clinical Trials Marktel S et al. Nature Medicine, 25: 234-241, 2019 Trials with GLOBE vector Jan 2019:0 Nine patients (3 adults, >18 yrs; 2 adolescent, 7-17 yrs; 4 paediatric, 3-7 yrs) treated Reduction in transfusion requirement in all 3 adult patients Three out of four evaluable paediatric participants discontinued transfusions; remained transfusion independent, with 24 months follow-up b-thalassaemia Gene Therapy : Clinical Trials Marktel S et al. Nature Medicine, 25: 234-241, 2019 Trials with GLOBE vector Jan 2019:0 Nine patients (3 adults, >18 yrs; 2 adolescent, 7-17 yrs; 4 paediatric, 3-7 yrs) treated Reduction in transfusion requirement in all 3 adult patients Three out of four evaluable paediatric participants discontinued transfusions; remained transfusion independent, with 24 months follow-up  bluebird bio inc led trials: BB305 vector Thompson AA et al. (2018) N Engl J Med. 378: 1479-1493 § 9 b-thal major (β0/β0 or low β+/β+) and 13 b-thal intermedia (non-β0/β0) genotypes § 13 patients with non-β0/β0 genotypes, median 26 months follow-up all but one discontinued transfusions; Hb 8.2–13.7 g/dL; HbAT87Q 3.4–10 g/dL § 9 patients with β0/β0 / low β+/β+ genotypes, reduced transfusion: median annualized transfusion volume decreased by 73%; transfusions discontinued in three patients Magrin E et al. (2022) Nat Med. 28: 81–88 Altered aspects of ex vivo gene therapy procedure 3 SCD patients; 42 month follow up; 2/3 positive results 4 (milder form) of b-thalassaemia; all no longer needed red blood cell transfusion; 72 months follow up Note: Efficacy needs >2 lentiviral vector copies per cell, or high residual g-globin or b-globin expression  Bluebird Bio SCD Gene Therapy Trial; BB305 vector § Single patient: βS/βS + aa/a- [Note: compound heterozygous SCD/a-thal] § Frequent sickling crises; transfusions plus iron chelation § Gene therapy at age 13 years in October 2014. 50% normal HbA restored from 9 months post-treatment Marked reduction in RBC sickling propensity at 12 months after treatment At 15 months after treatment: § Crisis-free § Correction of biological hallmarks (haematological parameters) § HOWEVER, failure to reproduce results on additional 7 patients Ribeil JA et al. (2017) N Engl J Med. 376: 848-855.  Kanter J et al. (2022) Results: BB305 vector 35 patients treated; 25 SCD patients evaluated; median follow up 17.3 month Average 40% of haemoglobin from therapy No vaso-occlusive crises after gene therapy  https://www.nature.com/articles/d41587-019-00026-3 § “Zynteglo” § Limited approval from EMA (June 2019) § Only for thalassaemia intermedia patients over 12 years old €1.6 million ($1.8 million) Zynteglo and Skysona (cerebral adrenoleukodystrophy gene therapy) approved in USA Zynteglo: $2.8 million Skysona: $3 million  Leber's congenital amaurosis (LCA) qLCA: degeneration of the retina. qCaused by mutations in the RPE65 gene: controls production of an enzyme responsible for the recycling of retinol, a chemical necessary for capturing light. qEarly-onset; complete loss of vision in early adulthood. qNo therapy available. Gene Therapy Procedure: In vivo delivery to retina of AAV vector with RPE65 gene Gene Therapy for Eyes: London Trial Long-term effect of gene therapy on Leber's congenital amaurosis. Bainbridge JW et al. (2015) N Engl J Med. 372: 1887-1897. HOWEVER, No clinical benefit even with higher doses of AVV vector … Gene therapy for Leber's congenital amaurosis is safe and effective through 1.5 years after vector administration. Simonelli F et al. (2010) Mol Ther. 18: 643-650. Russell S et al. (2017) Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65- mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet, 390: 849-860. FDA approves novel gene therapy to treat patients with a rare form of inherited vision loss Luxturna is the first gene therapy approved in the U.S. to target a disease caused by mutations in a specific gene https://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm589467.htm December 19, 2017 § AAV vector with RPE65 cDNA under CMV promoter control § Developed by Prof Jean Bennett and colleagues (University of Pennsylvania, USA) § Targeting LCA § Marketed by Spark Therapeutics Inc  Gene Therapy for Haemophilia B Haemophilia B: X-linked bleeding disorder Defect in coagulation factor IX (FIX) gene; serine protease that is critical for blood clotting Severe hemophilia B: less than 1% FIX; frequent bleeding episodes, crippling arthropathy and early death. “The Royal Disease“: Leopold Current treatment: Frequent intravenous injections of FIX protein concentrate (i.e., two to three times a week). Extremely expensive, and is associated with inhibitor (antibody) formation Gene Therapy for Haemophilia B Protocol Makris M (2018) Hemophilia gene therapy is effective and safe. Blood, 131: 952- 953.  Gene Therapy for Haemophilia B George LA et al. (2017) N Engl J Med. 377: 2215-2227. § AAV vector: liver-specific promoter; FIX Padua (FIX-R338L) transgene § Dose: 5×1011 vector genomes per kilogram § 10 men: FIX activity 2% or less of normal RESULTS: Vector-derived FIX coagulant activity sustained in all the participants FIX coagulant activity: 33.7±18.5% (range, 14 to 81) of normal Factor FIX use reduction Total of 8 of 10 participants did not use factor, and 9 of 10 did not have bleeds after vector administration. FDA Approves First Gene Therapy to Treat Adults with Hemophilia B https://www.fda.gov/news-events/press-announcements/fda-approves-first-gene-therapy-treat-adults-hemophilia-b November 22, 2022 U.S. Food and Drug Administration approved Hemgenix (etranacogene dezaparvovec), an adeno-associated virus vector-based gene therapy for the treatment of adults with Hemophilia B Human Gene Therapy Clinical Trials 1989-2021 Phases  Gene therapy medicines approved! €594,000 $475,000/ £282,000 $373,000 $425,000/eye Zynteglo June 2019 (EMA)/Sept 2022 (FDA) b-thal intermedia bluebird bio, USA $2,800,000 Zolgensma May 2019 FDA SMA Novartis, Switzerland $2,125,000 Skysona Sept 2022 FDA/EMA CALD bluebird bio, USA $3,000,000 Libmeldy Dec 2000 EMA MLD Orchard Therapeutics, UK £2,800,000 Hemgenix Nov 2022 FDA Haemophilia B CSL Behring, USA £3,500,000 Casgevy Nov 2023 HMRA (UK) SCD Vertex/CT, USA ? Ginn SL et al. (2018) J Gene Med. 20: e3015 USA approvals: https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/approved- cellular-and-gene-therapy-products  Gene Therapy – Summary Gene therapy offers great promise for treating currently intractable diseases and improving the therapy of others, especially when combined with gene delivery to stem cells. § Successes in clinical trials using viral vectors and CRISPR/Cas gene editing § Viral vector and CRISPR/Cas gene medicines approved Future advances to make gene therapy a widely applicable technology include: § In vivo targeted gene delivery systems § Minimise risks such as insertional mutagenesis § Gene editing correction § Reduce costs Gene therapy has finally reached true translational status (but currently very expensive)!  Appendix For student additional information  Lentiviral Vector Production: transient transfection of HEK293T cells Transfer plasmid: encodes therapeutic transgene Envelope plasmid: encodes virus envelope protein Packaging plasmid: encodes virus packaging proteins (Gag) and RNA genome replication enzyme (Pol) Adult Haemoglobin (HbA) 2 a-globin chains : 2 b-globin chain 4 haem groups  The a-globin and b-globin gene cluster and haemoglobin switching § Epsilon(e)-globin (HBE) and zeta(z)-globin (HBZ) genes expressed in embryo § Gamma(g)-globin (HBG) and alpha(a)-globin (HBA) genes expressed in foetus § Beta(b)-globin (HBB) and a- globin genes expressed post-natally § Always maintains 1:1 ration of a-type and b-type globin chains in resulting haemoglobin tetramer: z2e2; a2g2; a2b2 § Site of erythropoiesis also changes during development: § embryonic yolk sac; foetal liver; adult bone marrow § Note: locus control region (b- Higgs D et al., 2012 LCR) regulatory element  Repression of g-globin gene expression and the switch from g-globin to b-globin § KLF1 activates expression of the BCL11A (B cell CLL/lymphoma 11A) and LRF (leukaemia/lymphoma-related factor) genes [Note: LRF = zinc-finger and BTB-domain-containing 7A (ZBTB7A)] § BCL11A and LRF co-factor complexes bind to g-globin gene promoters § Note: NuRD histone deacetylase association Clinical Features of b-thalassaemia Anaemia - stimulation of erythropoietin production. Intense proliferation and expansion of bone marrow. Skeletal deformities. Abnormal RBCs -destroyed in the spleen -hepatosplenomegaly (splenectomy) Increased gastrointestinal (GI) tract iron absorption Emaciation and retarded growth Prognosis: ~85% with severe homozygous or compound heterozygous b-thal die by age of 5 due to severe anaemia Modulators of b-thalassaemia severity: HPFH Older Younger § Brothers: both inherited b- thalassaemia-major mutation § Younger brother has co- inherited HPFH mutation § High, persistent g-globin gene expression in younger brother compensates for lack of b- globin; less severe b- thalassaemia § Younger brother has more normal growth Polymerisation of HbS and “Sickling” of red Blood Cells Odievre MH et al. (2011) Indian J Med Res. 134: 532-537 Pathophysiology of Sickle Cell Disease Rees DC et al. (2010) Lancet 376: 2018-2031  Necrosis Induced by Repeated Sickle Crises Kidney failure Leg ulceration Humeral head Osteonecrosis and collapse Stroke Infarction and of multiple thoracic vertebral osteonecrosis end plates; "fish vertebrae"  Correction of SCD with CRISPR-Cas9 genome editing Homology directed repair of SCD mutation ~50% correction of SCD mutation in patient HSCs Gene correction Normal mRNA production Dever DP et al. (2016) Nature, 539: 384-389. SCD Gene Editing Correction Clinical Trials UC Consortium Launches First Clinical Trial Using CRISPR to Correct Gene Defect That Causes Sickle Cell Disease March 2021 https://www.ucsf.edu/news/2021/03/420137/uc-consortium-launches-first-clinical-trial- using-crispr-correct-gene-defect 4-year study; six adults and three adolescents with severe SCD Ex vivo electroporation of CRISPR-Cas + repair template DNA into patient HSCs Graphite Bio Announces U.S. FDA Fast Track Designation Granted to GPH101 for the Treatment of Sickle Cell Disease May 2022 https://www.biospace.com/article/releases/graphite-bio-announces-u-s-fda-fast-track- designation-granted-to-gph101-for-the-treatment-of-sickle-cell- disease/?keywords=Graphite+Bio GPH101 in the CEDAR trial (https://clinicaltrials.gov/ct2/show/NCT04819841), an open-label, multi-center Phase 1/2 clinical trial with 15 patients designed to assess the safety, engraftment success, gene correction rates, total hemoglobin, as well as other clinical and exploratory endpoints and pharmacodynamics in patients with severe SCD  SCD Gene Editing Correction Clinical Trials Serious Side Effect Sidelines Gene-Edited Sickle Cell Therapy from Graphite Bio Graphite Bio voluntarily paused a Phase 1/2 test of its gene-edited therapy for sickle cell disease. § First patient developed a “serious and unexpected adverse event” § Pancytopenia, decrease in blood cell counts. § Required treatment with blood transfusions and growth factors to boost cell counts. § Graphite Bio no longer expects to file an investigational new drug application for GPH102 in beta-thalassemia by mid-2024 https://medcitynews.com/2023/01/serious-side-effect-sidelines-gene-edited-sickle-cell- therapy-from-graphite-bio/ https://ir.graphitebio.com/press-releases/detail/84/graphite-bio-announces-voluntary- pause-of-phase-12-cedar Demyelinating diseases: ALD, MLD Poletti V and Biffi A (2019) Gene-Based Approaches to Inherited Neurometabolic Diseases. Hum Gene Ther. 30: 1222-1235 Mallack EJ, Turk B, Yan H, Eichler FS (2019) The Landscape of Hematopoietic Stem Cell Transplant and Gene Therapy for X- Linked Adrenoleukodystrophy. Curr Treat Options Neurol. 21: 61.  Gene Therapy for X-Linked Adrenoleukodystrophy (ALD) First to use Lentiviral Vector for Inherited Disorder § X-linked recessive inherited defect Deficiency in ALD protein; adenosine triphosphate-binding cassette transporter encoded by ABCD1 Severe brain demyelinating disease in boys Progression halted by allogeneic hematopoietic stem cell transplantation § Gene Therapy Approach ABCD1 cDNA into lentiviral vector: HIV-1–derived lentiviral vector (CG1711 hALD) expressing wild-type ABCD1 cDNA under the control of the MND (myeloproliferative sarcoma virus) promoter/enhancer Ex vivo infection of HSC + full myeloablative conditioning Reinfusion into original donor patients Cartier N et al. (2009) Science, 326: 818-823.  Gene Therapy for X-Linked Adrenoleukodystrophy (ALD) §Outcomes §2 patients treated 24 to 30 months of follow-up §Reconstitution of all blood lineages §Beginning 14 to 16 months after treatment: Progressive cerebral demyelination in the two patients stopped Clinical outcome comparable to that achieved by allogeneic HCT  Hematopoietic Stem-Cell Gene Therapy for Cerebral Adrenoleukodystrophy Eichler F et al. (2017) N Engl J Med. 377: 1630-1638. § 17 boys (4-13 years); cerebral X-ALD § Lenti-D vector; HSC/ex vivo § Median follow-up 29.4 months (range, 21.6 to 42.0) § No treatment-related death or graft-versus-host disease § 15 of the 17 patients (88%) were alive and free of major functional disability, with minimal clinical symptoms. Gene Therapy for Metachromatic Leukodystrophy (MLD) Metachromatic leukodystrophy (MLD): §Autosomal recessive §Lysosomal storage disease §Mutations in ARSA gene; deficiency of arylsulfatase A §Accumulation of the enzyme substrate sulfatide: oligodendrocytes, microglia, and certain neurons of the central nervous system, Schwann cells and macrophages of the peripheral nervous system §Sulfatide biuld-up: widespread demyelination and neurodegeneration; severe progressive motor and cognitive impairment. §Fatal in few years from disease onset  MLD Gene Therapy Biffi A et al. (2013) Science, 341: 1233158. §pCCLsin.cPPT.hPGK.hARSA.WPREmut6 - PGK.ARSA.LV §Ex vivo bone marrow HSC §Full myeloablation conditioning; busulfan §9 patients; 6 infants, 3 older §Stable engraftment; polyclonal, all lineages §Stable ARSA enzyme activity, inc. CNS §Decline in periferal neuropathy (proportional to level of engraftment) §Motor ability: rescued totally over time §Normal for age cognitive ability EU Approves MLD Gene Therapy “Libmeldy” (Dec 17 2020) https://www.ema.europa.eu/en/medi cines/human/EPAR/libmeldy Joe Elson, 11 years old, 7 years after gene therapy Sister Connie to receive treatment List price: £2.8 million NHS stuck a confidential deal!