WELCOME AND INTRODUCTION Perry Shieh, MD, PhD

WELCOME AND INTRODUCTION Perry Shieh, MD, PhD
Associate Professor of Neurology, University of California, Los Angeles, USA This meeting is not part of the AAN Annual Meeting official programming and no CME is given for attendance. This Industry Therapeutic Update is sponsored and organized by AveXis.. © AveXis, Inc. All Rights Reserved MED-CON-UNB US 05/2019

DISCLOSURES Perry Shieh, MD, PhD Alexion: Speaking AveXis: Consulting
Biogen: Consulting and speaking CSL Behring: Speaking Grifols: Speaking Sarepta: Consulting

INDUSTRY THERAPEUTIC UPDATE OBJECTIVES
Explore the progress and current clinical landscape of gene therapy (GT) Understand the fundamentals of GT including the potential application of GT for neuromuscular disorders Discuss the translation of GT into clinical practice, including the safe handling of vectors

FACULTY INTRODUCTIONS
Perry Shieh, MD, PhD Associate Professor of Neurology, University of California, Los Angeles, USA Meredith Schultz, MD Medical Director, Translational Medicine, AveXis Inc., USA Christopher Walker, PhD Professor of Pediatrics, Center for Vaccines and Immunity, The Research Institute, Nationwide Children’s Hospital, Columbus, Ohio, USA Chris Jenkins, PhD, MPH, RBP, CHMMMD Principal Partner & Chief Gene Therapy Biosafety Officer, Clinical Biosafety Services, USA

AGENDA Time Presentation Title Chair/Speaker 7:30 – 7:40 PM
Welcome and introduction Perry Shieh, MD, PhD (Chair) 7:40 – 8:00 PM Gene therapies: a new frontier for neuromuscular disorders Meredith Schultz, MD 8:00 – 8:20 PM A focus on adeno-associated virus (AAV) antibodies Christopher Walker, PhD 8:20 – 8:40 PM Biosafety considerations for the clinical use of AAVs Chris Jenkins, PhD 8:40 – 8:50 PM Panel discussion All Speakers Facilitated by Chair 8:50 – 9:00 PM Questions and answers 9:00 – 9:05 PM Closing remarks

? HOUSEKEEPING Please turn off your mobile devices
If you require any assistance, please notify the staff Voting keypads can be found on the table in front of you, please share your feedback with us when questions are asked throughout the meeting There will be time for questions at the end of the Industry Therapeutic Update.

EVALUATION QUESTIONS Please kindly submit your response to this question using the voting keypads provided Question 1: Please select your area of specialty from the options provided below: 1 = General Neurologist 2 = Geneticist 3 = Neuromuscular Disorder Specialist 4 = Neuromuscular Physical Therapist 5 = Other Neurology Sub-Specialist 6 = Pediatric Neurologist 7 = Researcher 8 = Other

EVALUATION QUESTIONS Please kindly submit your response to this question using the voting keypads provided Question 2: How would you describe your current level of knowledge on gene therapy and its use in treating neuromuscular disorders? 1 = Very high 2 = High 3 = Moderate 4 = Low 5 = Very low

NEUROMUSCULAR DISORDERS: AN OVERVIEW
Hereditary paraplegias Distal myopathies Motor neuron diseases Congenital myasthenic syndromes Ion channel muscle diseases Malignant hyperthermia Congenital myopathies Other neuromuscular disorders Neuromuscular disorders Other myopathies Metabolic myopathies Myotonic syndromes Hereditary cardiomyopathies Muscular dystrophies Hereditary ataxia Congenital muscular dystrophies Hereditary motor and sensory neuropathies NMDs are a heterogeneous group of conditions affecting the anterior horn motor cells, peripheral nerves, neuromuscular junctions, or muscles1 Can be inherited or acquired1, and affect approximately 1 in 1,000 people worldwide2 Management of NMDs can include supportive care3, physical therapy1, symptom treatment4 and, where available, targeted treatment5 NMD; Neuromuscular disorders 1. Demir, Y. (2017) Neuromuscular Diseases and Rehabilitation. Neurological Physical Therapy. Chapter Zatz. M et al. (2016) Neuromuscular disorders: genes, genetic counselling and therapeutic trials. Genet Mol Bol. 39(3): ; 3. Hughes, R et al. (2005) Supportive care for patients with Guillain-Barre Syndrome. Arch Neurol. 62(8):1194– Gilhus, N et al. (2011) Myasthenia Gravis: A Review of Available Treatment Approaches. Autoimmune Diseases. Article ID ; 5. Jirka, S et al. (2015) An update on RNA-targeting therapies for neuromuscular disorders. Curr Opin Neurol 28(5): 1. Demir, Y. (2017) Neuromuscular Diseases and Rehabilitation. Neurological Physical Therapy. Chapter 11 2. Zatz. M et al. (2016) Neuromuscular disorders: genes, genetic counselling and therapeutic trials. Genet Mol Bol. 39(3): 3. Hughes, R et al. (2005) Supportive care for patients with Guillain-Barre Syndrome. Arch Neurol. 62(8):1194–1198 4. Gilhus, N et al. (2011) Myasthenia Gravis: A Review of Available Treatment Approaches. Autoimmune Diseases. Article ID 5. Jirka, S et al. (2015) An update on RNA-targeting therapies for neuromuscular disorders. Curr Opin Neurol 28(5):515-21 6. Aguti, S et al. (2018) The progress of AAV- mediated gene therapy in neuromuscular disorders, Expert Opinion on Biological Therapy, 18(6): 7. Duan, D et al. (2018) Systemic AAV Micro-dystrophin Gene Therapy for Duchenne Muscular Dystrophy. Molecular Therapy Review. 26(10):

NEUROMUSCULAR DISORDERS: AN OVERVIEW
A range of conditions affect the functioning of muscles1 Broad range of causes and symptoms1 Paraesthesias and numbness Loss of dermal sensation1 Fasciculations Involuntary muscle contractions2 Muscular atrophy and weakness Loss of muscle bulk and reduced muscle strength2 1. Nayak, R. (2017) Practical approach to the patient with acute neuromuscular weakness. WJCC. 5(7): 2. McDonald, C. (2012) Clinical approach to the diagnostic evaluation of hereditary and acquired neuromuscular diseases. Phys Med Rehabil Clin N Am. 23(3):

GENE THERAPY: A NOVEL TREATMENT OPTION
Gene therapy is a novel technique fast becoming a key treatment option for human genetic diseases1 Recent advancements in gene therapy strategies are beginning to achieve clinical successes for monogenic diseases1 Developments in gene delivery techniques have improved safety and efficacy2,3 Number of Gene Therapy Clinical Trials Approved Worldwide 1989 – 2018 Total patients treated to date: 2926 Updated December 2018 Now, viral gene therapy products have achieved regulatory approval in the US and EU4–7 with over 2,600 trials ongoing or completed worldwide8 1. Kumar SRP et al. Mol Ther Methods Clin Dev. 2016; 3: 16034; 2. Naso MF. BioDrugs 2017;31:317–334; 3. Thomas CE, et al. Nat Rev Genet 2003;4(5):346–358; 4. Hoggatt J. Cell 2016;166(2):263; 5. accessed October 2018; 6. accessed October 2018; 7. accessed May 2019; 8. Ginn, S et al. Gene therapy trials worldwide in 2017: an update. J Gene Med. 20(5):e3015 Image adapted from: The Journal of Gene Medicine, 2018. Available at: accessed May 2019.

GENE THERAPY: THE CHALLENGES
There are several technical challenges that must be overcome to develop optimal vectors and viable gene therapies for neuromuscular disorders These can be broadly categorized as issues with1–7: Gene Delivery For CNS Disorders, Crossing the BBB Durability Minimization of Immune Responses Safety Concerns Practicalities of Gene Therapy CNS; Central nervous system, BBB; Blood brain barrier 1. Learn.Genetics. Challenges in Gene Therapy. Available at: accessed February 13, Kay MA. Nat Rev Genet 2011;12(5):316–328; 3. Wang D, Gao G. Discov Med 2014;18:67–77; 4. Basner-Tschakarjan E, Mingozzi F. Front Immunol 2014;5:350; 5. Williams DA, Thrasher AJ. Stem Cells Transl Med 2014;3(5):636–642; 6. BioProcess Online. Overcoming The Manufacturing Hurdles Of Cell & Gene Therapy. Available at: accessed February 13, Foust, K. et al. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes in the CNS. Nat Biotechnol. 2009;27(1)59-65

GENE THERAPIES: A NEW FRONTIER FOR NEUROMUSCULAR DISORDERS
Meredith Schultz, MD Medical Director, Translational Medicine, AveXis Inc., USA This meeting is not part of the AAN Annual Meeting official programming and no CME is given for attendance. This Industry Therapeutic Update is sponsored and organized by AveXis. © 2019 AveXis, Inc. All Rights Reserved MED-CON-UNB US 05/2019

DISCLOSURES Meredith Schultz, MD AveXis employee
Previously a principal investigator for AveXis clinical trials

SESSION OBJECTIVES Briefly review the history and progress of gene therapies to date Consider the outlook for gene therapy (GT) in the context of neuromuscular disorders Discuss the role of adeno-associated virus (AAV) vectors as viable delivery candidates in GT

HISTORY OF GENE THERAPY AND APPLICATIONS IN NEUROMUSCULAR DISORDERS

FROM CONCEPT TO CLINIC: GENE THERAPY HISTORY
First therapeutic gene transfer in ADA patients2 China first country to approve a gene therapy-based product for clinical use4 Europe approves GSK to treat patients with ADA-SCID6 US approves axicabtagene ciloleucel (CAR-T cell therapy) for large B-cell lymphoma8 2017 1989 1990 1999 2003 2009 2016 Aug Dec Oct First officially approved gene transfer into humans (Steven A. Rosenberg)1 Gene therapy clinical trial patient Jesse Gelsinger, fatal immune response3 First successful phase 3 gene therapy clinical trial in the EU5 US approves tisagenlecleucel (CAR-T cell therapy) for leukemia7 US approves first directly administered gene therapy, voretigene neparvovec-rzyl for retinal dystrophy9 ADA-SCID, severe combined immunodeficiency due to adenosine deaminase deficiency. CAR, chimeric antigen receptor. Timeline adapted from Wirth T, et al. Gene 2013;525:162–169; 1. Rosenberg SA, et al. N Engl J Med 1990;323(9):570– Blaese RM, et al. Science 1995;270(5235):475– Sibbald B. CMAJ 2001;164(11): accessed October 2018; 5. Wirth T, et al. Gene 2013;525:162– Hoggatt J. Cell 2016;166(2): accessed October accessed October accessed October 2018.

GENE THERAPY IS A KEY TREATMENT OPTION FOR MONOGENIC DISEASES INCLUDING NEUROMUSCULAR DISORDERS
GT shows promise in targeting monogenic diseases1 >10,000 human genetic diseases are monogenic2, examples include: Blood Disorders i.e. Thalassemias, Sickle Cell Anemia, Hemophilia2 Ophthalmic Disorders i.e. Inherited Retinal Diseases/Dystrophy3 Neurological Disorders i.e. Rett Syndrome4 Neurodegenerative Diseases i.e. Huntington's Disease,2 SMA,5 ALS6 and DMD7 ALS, Amyotrophic lateral sclerosis; DMD, Duchenne Muscular Dystrophy; GT, gene therapy; SMA, Spinal muscular atrophy 1. Boudes PF. Eur J Intern Med 2014;25(1):31–36; 2. accessed March 2019; 3. Bok D. Arch Ophthalmol. 2007;125(2):160–164; 4. Liyanage VRB & Rastegar M. Neuromolecular Med 2014;16(2);231–264; 5. Prior TW. Genet Med 2010;12(3):145–152; 6. Bosco DA. Nature Education 2015;8(3):4; 7. Alameddine H, et al. Neuromuscular Disord. 1994;4(3):

THERE ARE 4 KEY APPROACHES TO GENE THERAPY1,2
Gene replacement Gene addition Gene replacement mutant gene functional gene therapeutic gene Replacing a mutated gene that causes disease with a healthy gene Genetic factors and/or environmental factors replacement addition Introduce a new gene into the body often to supplement a targeted therapeutic agent Gene addition disease correction disease alleviation Gene inhibition Gene editing mutant gene gene silencer mutant gene gene corrector Gene inhibition Inactivating a mutated gene that is over-producing its product by targeting RNA editing knockdown Making a targeted change to the gene sequence Gene editing disease correction disease correction Diagram adapted from Wang D, et al. Discov Med 2014;18:151–161. 1. accessed October Wang D, et al. Discov Med 2014;18:151–161.

ESSENTIAL COMPONENTS OF GENE DELIVERY
Transgene expression cassette1 Three key components:1 Vector Vehicle by which a transgene is delivered to the targeted cells Transgene cDNA coding the replacement gene Promoter “Turn on” switch to modulate expression of the transgene Promoter Transgene scAAV ITR DNA Vector Viral Vector Also typically includes, either: Termination signal1 To end gene transcription ITRs2 At either end of the cassette to allow for synthesis of cDNA Diagram adapted from Wang D, et al. Discov Med 2014;18:151–161. cDNA, complementary DNA; ITRs, inverted terminal repeats. Wang D, et al. Discov Med 2014;18:67– Ayuso E, et al. Curr Gene Ther 2010;10:423–436.

ROLE OF ADENOVIRUS-ASSOCIATED VECTORS (AAV) IN THE DELIVERY OF GENE THERAPIES

CLINICAL TRIAL DATA – VECTORS AND GENES
Vectors used in gene therapy clinical trials Gene types transferred in gene therapy clinical trials accessed 12th April 2019 accessed 12th April 2019 References:

FIVE MAIN TYPES OF VIRAL VECTORS STUDIED
The AAV vector has been designed to be non-pathogenic and non-inflammatory1 Vector Genetic material Packaging capacity Tropism Inflammatory potential Safety Main limitations Main advantages Retrovirus RNA 8 kb Dividing cells only Low Oncogenic potential Only transduces dividing cells; integration might induce oncogenesis in some applications Persistent gene transfer in dividing cells Lentivirus Broad Integration might induce oncogenesis in some applications Persistent gene transfer in most tissues HSV-1 dsDNA 40 kb* 150kb† Strong for neurons High Immunogenic Inflammatory; transient transgene expression in cells other than neurons Large packaging capacity; strong tropism for neurons Adenovirus 8 kb* 30kb‡ Broad Highly immunogenic Capsid mediates a potent inflammatory response Extremely efficient transduction of most tissues AAV ssDNA§ <5 kb Broad, with the possible exception of hematopoietic cells Low Generally non-pathogenic Small packaging capacity limits the range of therapeutic genes that can be used in this system Low- or non-inflammatory; non-pathogenic *Replication defective. †Amplicon. ‡Helper dependent. §The inclusion of inverted terminal repeats (ITRs) at either end of the cassette allows for synthesis of complementary DNA and thus produces dsDNA AAV, adeno-associated virus; dsDNA, double-stranded DNA; HSV-1, herpes simplex virus-1; ssDNA, single- stranded DNA. 1. Thomas CE, et al. Nat Rev Genet 2003;4(5):346–358.

AAV: A PROMISING VECTOR SYSTEM FOR GENE REPLACEMENT1
Transduction Can be engineered for selective cell targeting and optimized transduction1 Safety Non-pathogenic; designed to not integrate the transgene into the host genome2 Less immunogenic than other viruses1 Tropism AAV serotypes display broad tropisms across the serotypes identified3,4 rAAV9 model (cross section) Versatility AAVs can be engineered for specific functionality in gene therapy applications1 Illustration adapted from DiMattia. J Virol 2012;86:6947. AAV, adeno-Associated Virus; rAAVs, recombinant AAVs. AAV10 serotype isolated from rhesus macaques; 1. Naso MF. BioDrugs 2017;31:317–334; 2. Thomas CE, et al. Nat Rev Genet 2003;4(5):346–358; 3. DiMattia MA, et al. J Virol 2012;86:6947–6958; 4. Hammond SL, et al. PLoS One 2017;12:e

AAV IS A PROMISING VECTOR SYSTEM FOR GENE REPLACEMENT1
Constructing an AAV Vector AAV viral vectors are made by: Deleting the natural viral genes to make them replication incompetent and replacing with a “transgene cassette” Structure of Wild-type Virus Genome ITR Replication Genes Capsid Genes Rep Cap AAV virus ❌ ❌ Vector Construct Transgene Cassette ITR P T Promoter Terminator Sequences placed between the inverted terminal repeats (ITRs) include a promoter, the gene of interest, and a termination signal ITRs give the transgene the ability to form self- complementary molecule AAV vector AAV, adeno-Associated Virus ; ITR, Diagram adapted from Wang & Gao. Discov Med. 2014;18(97):67–77. 1. Naso MF, et al. BioDrugs 2017;31:317–334.

AAVS DELIVER GENES WITHOUT GENOMIC INTEGRATION1
AAV binds to cell nucleus; releases contents Nucleus AAV taken into cell via endosome DNA forms circular episome Protein expressed Endosome breaks down Endosome Inert AAV vector Therapeutic DNA Outside body Inside body 1 The vector is taken into the cell via the endosome1 2 The endosome breaks down2 3 Therapeutic DNA enters the cell nucleus1 4 The DNA forms a circular episome1 The episome folds upon itself to form a self-complementary double stranded DNA molecule ready for transcription3 5 The resulting transcript leaves the nucleus and travels to the ribosome for translation (protein synthesis)4 1. Naso MF, et al. BioDrugs 2017;31:317–334; 2. Xiao PJ, et al. J Virol2012;86:10462–10473 3. Mern DS, et al. PLoS One 2017;12:e ; 4. Wang J, et al. Proc Natl Acad Sci USA 2007;104:13104–1 Adapted from Akst J. The Scientist. June 2012.

TRANSLATION, TRANSCRIPTION, TRANSDUCTION
1 Transduction 2 Transcription 3 Translation A T G C Viral vector T A C G DNA A T G C RNA Transgene protein A U G C DNA mRNA Transgene DNA T A C G Delivery of transgene into cell mRNA synthesis Protein synthesis Adapted from

Viral vector platforms Transgene persistence
TRANSGENES DELIVERED VIA AAV VECTORS ARE ABLE TO ACHIEVE DURABLE EFFECT Disease pathogenesis Viral vector platforms Primary target Transgene persistence Parkinson disease Complex AAV Neurons Non-human primates: 15 ya,b,1 Humans: 4 yb,2 Hemophilia B Monogenic Liver Non-human primates: 5.5 ya,b,3 Dogs: 4.5 ya,4; 8 yb,5 Humans: 4 ya,6; 10 yb,7 Inherited retinal disease Retinal pigment epithelial cells Dogs: 11 ya,8 Humans: 3 ya,9 SMA Motor neurons Mice: >250 daysa,10 Humans: up to ~5 ya,11 aPersistence of treatment effect. b Transgene persistence determined by presence in tissues; SMA, Spinal Muscular Atrophy 1. Sehara Y, et al. Hum Gene Ther Clin Dev 2017;28: Bartus RT, et al. Neurobiol Dis 2015;78:162– Nathwani AC, et al. Mol Ther 2011;19:876– Callan MB, et al. PLoS One 2016;11:e ; 5. Niemeyer GP, et al. Blood 2009;113(4):797– Nathwani AC, et al. N Engl J Med 2014;371:1994– Buchlis G, et al. Blood 2012; 119:3038– Cideciyan AV, et al. Proc Natl Acad Sci USA 2013;110:E517–E Bennett J, et al. Lancet 2016;388:661– Foust KD, et al. Nat Biotechnol 2010;28:271– Mendell J, et al. 22nd Annual SMA Researcher Meeting, Dallas, June 14–16, Poster #4.

DESIGN OF GENE THERAPY TREATMENTS AND THEIR CLINICAL DEVELOPMENT ARE ADVANCING RAPIDLY1,2
Viral Vector Platform Disease Clinical Study Non-integrating viral vectors Retrovirus γ-retrovirus Adenosine deaminase severe combined immunodeficiency (“bubble baby disease”) Approved in EU (2016) Lentivirus Transfusion dependent ß-thalassemia Phase 3 (2016) Adrenoleukodystrophy (caused by ABCD1 gene) Phase 2/3 (2013) AAV1 Lipoprotein lipase deficiency Approved in EU (2012) AAV2 Inherited retinal disease Approved in US (2017) AAV5 Hemophilia B Phase 1/2 (2018) AAV8 Hemophilia A AAV9 Spinal muscular atrophy Phase 3 (2017) Limb girdle muscular dystrophy type 2D Phase 1 (2015) AAV1/2/8 and 9 Pompe Disease Phase 1/2 (2018 and 2019) Duchenne Muscular Dystrophy Phase 1/2 (2017) Provided by presenter for CureSMA Integrating viral vectors Non-integrating viral vectors AAV, Adeno-associated virus 1. Kumar SRP et al. Mol Ther Methods Clin Dev. 2016; 3: 16034; 2. (accessed May 2019) Original table

OVERCOMING THE BBB: AAV9 TARGETS MOTOR NEURONS1
Most therapies do not cross the BBB: The BBB and extensive brain vasculature pose obstacles to GT development for neurological diseases In 2009, a seminal discovery changed how we deliver gene therapies to the brain One-time administration of AAV9-GFP to the bloodstream led to robust expression in cells throughout the brain and spinal cord AAV9 delivered into an adult mouse via a tail vein injection BBB, blood-brain barrier; GT, Gene Therapy; GFP, green fluorescent protein Foust KD, et al. Nat Biotechnol 2009;27(1):59–65.

QUESTIONS Please kindly submit your response to this question using the voting keypads provided Question 1: In your opinion, aside from overall survival, what would be the most important outcome of gene therapy for neuromuscular disorders in clinical practice? 1 = Restoration of muscle function 2 = Arresting decline of muscle function 3 = Improvement in quality of life for the patient/caregiver 4 = Reduced need for long-term treatment interventions

QUESTIONS Please kindly submit your response to this question using the voting keypads provided Question 2: In your view, what is the highest priority for research into gene therapy for neuromuscular disorders? 1 = Application of modified AAV vectors to the widest possible range of neuromuscular disorders 2 = Establishing the efficacy of the investigational therapies 3 = Establishing the safety of the investigational therapies 4 = Increasing our depth of knowledge about neuromuscular disorders

CONCLUSIONS GT is a novel technique1 and some products have already achieved regulatory approval in the US and EU2–5 GT aims to provide sufficient gene expression in enough targeted cells to ameliorate or correct a dysfunctional phenotype6 GT has shown promise in the treatment of various diseases, with many trials currently in place7 Developments in gene delivery techniques have shown AAVs to be one of the most promising and versatile vectors for gene transfer and expression8 With lower risk of inflammation and immunogenicity, AAVs are one of the most promising vectors for gene transfer and expression in monogenic disorders8,9 Based on AAV9’s capacity to cross the BBB, investigational gene therapies are showing promise for the treatment of neuromuscular disorders10,11 GT, Gene Therapy; AAV, Adeno Associated Virus; BBB, blood-brain barrier 1. Kumar SRP et al. Mol Ther Methods Clin Dev. 2016; 3: 16034; 2. Hoggatt J. Cell 2016;166(2):263; 3. accessed October 2018; accessed October 2018; 5. accessed October 2018; 6. Collins M, et al. Proc R Soc B 2015;282: ; Naso MF. BioDrugs 2017;31:317–334; 9. Thomas CE, et al. Nat Rev Genet 2003;4(5):346– Foust KD, et al. Nat Biotechnol 2009;27(1):59–65; 11. Mendell JR et al. NEJM (18):

A FOCUS ON ADENO-ASSOCIATED VIRUS (AAV) ANTIBODIES
Christopher Walker, PhD Professor of Pediatrics, Center for Vaccines and Immunity, The Research Institute, Nationwide Children’s Hospital, Columbus, Ohio, USA Not an official event of the 2019 American Academy of Neurology (AAN) Annual Meeting. Not sponsored, endorsed, or accredited by AAN. This Industry Therapeutic Update is sponsored and organized by AveXis. ©2019 AveXis, Inc. All Rights Reserved MED-CON-UNB US 05/2019

DISCLOSURES Christopher Walker, PhD Consultant for AveXis

SESSION OBJECTIVES Review the structure of the AAV capsid and its recognition by neutralizing antibodies Discuss the impact of anti-AAV antibodies on gene therapy and strategies for immune response avoidance Examine the measurement, prevalence and timing of anti-AAV antibody responses AAV, adeno-associated virus

IMMUNE RECOGNITION OF AAV VECTORS AND VECTOR TREATED CELLS
Antibody CD8* T cell TLR2 TCR MHC class I AAV TLR9 Endosome Proteasome Peptides Endoplasmic reticulum Nucleus 3’ 5’ CD8+ T cell targeting of transduced cells Capsid degradation and MHC presentation Generation of transgene-specific antibodies Activation of TLRs and innate immunity NAbs Golgi Neutralizing antibodies can block the AAV capsid:1 Attachment to cellular receptor(s) Uncoating of the AAV particle after entry into a target cell Image adapted from: Wang D, et al. Nat Rev Drug Discov Feb 1 AAV, adeno-associated virus; TCR, T cell receptor; TLR, toll-like receptor; MHC, major histocompatibility complex; NAbs, neutralizing antibodies 1. Wang D, et al. Nat Rev Drug Discov Feb 1 Focus on attachment and entry step highlighted in red dotted box.

ANTI-AAV ANTIBODIES AND AVOIDANCE STRATEGIES

ANTIBODIES TARGETING THE AAV CAPSID ARE A CRITICAL FACTOR IN THE OUTCOME OF AAV GENE DELIVERY
The AAV capsid is comprised of 3 proteins designated VP1, VP2, and VP31 Natural infection with AAV elicits antibodies against the capsid proteins that can interfere with AAV-mediated gene delivery1 VP, Viral Protein; AAV, Adeno Associated Virus 1. Giles AR, et al. J Virol. 2018;92(20);e Left. Cryo-EM reconstruction of AAV9 capsid (fuschia) in complex with a mouse neutralizing monoclonal antibody Fab FAB (blue). Right Cross-section of the AAV9-antibody Fab complex to illustrate the challenge presented by neutralizing antibodies..

STRATEGIES TO AVOID NEUTRALIZING ANTIBODIES
The impact of anti-AAV antibodies is blunted when vectors are delivered directly to tissues (for instance, muscle) or an immune privileged site (for instance, eye)1 Systemic vector delivery remains challenging1 Experimental (pre-clinical) strategies to avoid anti-AAV antibodies include: (i) Plasmapheresis and saline flush to lower antibody titers and provide a temporary window for AAV vector delivery1 (ii) Reduction in circulating B cells prior to dosing2 (iii) Engineering of the AAV capsid to eliminate neutralizing antibody epitopes3 AAV, adeno-associated virus 1. Mingozzi F and High KA. Blood. 2013;122(1): Corti Met al .Mol Ther Methods Clin Dev Bartel M, et al. Front Microbiol. 2011;2:204.

IMPLICATIONS OF ANTI-AAV ANTIBODIES FOR TRIALS AND GENE THERAPY
FOR NOW, TREATMENT FAILURE IS AVOIDED ONLY BY EXCLUSION OF PATIENTS WITH ANTI-AAV ANTIBODIES THAT EXCEED A LOW THRESHOLD TITER1 AAV, adeno-associated virus 1. Vandamme C, et al. Hum Gene Ther. 2017;28(11):

PREVALENCE OF ANTI-AAV NEUTRALIZING ANTIBODIES

WHAT IS THE PREVALENCE OF ANTI-AAV ANTIBODIES IN HUMAN POPULATIONS?
At least 13 AAV capsid serotypes have been identified in humans and non-human primates1 Some factors to consider when assessing neutralizing antibodies: Capsid serotypes determine tissue tropism2,3 and susceptibility to antibody neutralization Only a small number of human (AAV1, AAV2, AAV8, AAV9)1,4,5 and non-human primate (AAVrh10, AAVrh74)6 capsids have been developed for clinical use to date AAV, adeno-associated virus 1. Büning H, et al. Mol Ther. 2015;23(11): Wu Z, et al. Mol Ther. 2006;14(3): Gurda BL, et al. J Virol. 2013;87(16): Kumar SRP et al. Mol Ther Methods Clin Dev. 2016; 3: Naidoo J, et al. Mol Ther. 2018;26(10): György B, et al. Mol Ther Methods Clin Dev. 2018;13:1-13.

MEASURING SERUM ANTI-AAV ANTIBODY TITERS
Serum antibodies that bind the AAV capsid can be measured by ELISA or neutralization assays1 Substrate For clinically relevant AAV serotypes, anti-capsid ELISA antibody titers are roughly equivalent to neutralization titers2 Detection antibody (conjugated) Serum antibodies ELISA is far less costly and difficult to perform and easier to standardize than neutralization assays1,3 AAV capsid AAV, adeno-associated virus; ELISA, enzyme-linked immunosorbent assay 1. Rincon MY, et al. JMIR Res Protoc. 2016;5(2):e Martino AT, et al. Methods Mol Biol. 2011;807: Meliani A, et al. Hum Gene Ther Methods. 2015;26(2):45-53.

CLINICAL STUDIES WITH INTRAVENOUSLY ADMINISTERED AAV GENE THERAPY
Clinical Study1 Vector Route/Doses Anti-AAV Cutoff SMA1, Phase 1/2 AAV9 Intravenous; 6x x1014 vg/kg <1:50 IgG Hemophilia A, AAV5 Intravenous; 6x x1013 vg/kg <1:1 NAb SPK-8011, rAAV Intravenous; 5x x1012 vg/kg Hemophilia B, Phase 2 AMT-060, Intravenous; 2x1013 vg/kg DMD, Phase 1 AAVrh74 Intravenous; 2x1014 vg/kg <1:400 IgG AAV, adeno-associated virus; SMA, Spinal Muscular Atrophy; DMD, Duchenne Muscular Dystrophy 1. S. Al-Zaidy, From Clinical Trials to Clinical Practice: Practical Considerations for Gene Replacement Therapy in SMA Type 1 (Manuscript in Preparation)

AAV PREVALENCE BY SEROTYPE AND TITER
Prevalence of neutralizing factors in serum against AAV types 1, 2, 5, 6, 8, and 9 Percentages of donors seropositive for neutralizing factors specific to AAV types 1, 2, 5, 6, 8, and 9* Distribution of neutralizing factor titers to AAV types 1, 2, 5, 6, 8, and 9** 70 120 60 100 50 80 40 Seropositive individuals (%) Distribution of titers (%) 60 30 40 20 10 20 AAV1 AAV2 AAV5 AAV6 AAV8 AAV9 AAV1 AAV2 AAV5 AAV6 AAV8 AAV9 n: 152 89 49 56 50 62 1:20 1:200 ≥1:400 Boutin et al. Human Gene Therapy :704–712 *Sera were judged positive for neutralizing capacity when a 1:20 dilution of serum inhibited vector transduction by 50% or more **The percentage of total neutralizing factor titers is shown for all the seropositive samples obtained

ANTI-AAV ANTIBODY PREVALENCE BY AGE AND SEROTYPE
Healthy Controls MPS IIIA 80 Age 2–7 Age >8 80 Age 2–7 Age >8 60 60 Ab-positive (%) 40 Ab-positive (%) 40 20 20 1 2 3 4 5 6 7 8 9 rh74 1 2 3 4 5 6 7 8 9 rh74 AAV serotypes AAV serotypes Data illustrate the potential association of AAV seroprevalence with age and disease conditions1 AAV, adeno-associated virus; MPS, mucopolysaccharidosis 1. Fu H, et al. Hum Gene Ther Clin Dev. 2017;28(4):

ANTI-AAV NEUTRALIZING ANTIBODIES BY AGE
Healthy Controls1 Hemophilia Patients1 AAV1 AAV2 AAV3 AAV4 AAV5 AAV1 AAV2 AAV3 AAV4 AAV5 100 100 80 80 60 60 Percentage Percentage 40 40 20 20 Birth year 1991–2000 1981–1990 1971–1980 1961–1970 –1960 Birth year 1991–2000 1981–1990 1971–1980 1961–1970 –1960 Age (years) 12–21 22–31 32–41 42–51 ≥52 Age (years) 12–21 22–31 32–41 42–51 ≥52 (n=21) (n=15) (n=15) (n=8) (n=26) (n=10) (n=16) (n=14) (n=8) (n=11) (Japan cohort, healthy controls) AAV, adeno-associated virus 1. Mimuro J, et al. J Med Virol. 2014;86(11):

NEUTRALIZING ANTIBODY PREVALENCE AGAINST AAV2 AND AAV8 CAPSIDS IN CHILDREN
NAb, % positive with titer ≥ 1:401 50 AAV2 AAV8 40 30 20 10 0–2 3–6 7–11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Age (months) Age (years) AAV, adeno-associated virus; NAb, neutralizing antibody 1. Calcedo R et al. Clin Vaccine Immunol Sep; 18(9): 1586–1588

BASELINE ANTI-AAV ANTIBODY TITERS OBSERVED IN INFANTS ENROLLED IN A PHASE 3 CLINICAL TRIAL
Subject AAV9 Antibody Titer Maternal* Patient Enrolled 1 <1:12.5 2 3 4 nc 1:12.5 5 6 7 1:100 8 1:25 9 10 11 12 13 14 15 16 17 18 19 20 1:50 21 22 Not enrolled 23 24 25 Incidence of anti-AAV9 antibodies was low in infants (aged <6 months) screened for enrollment in a phase 3 clinical trial of gene therapy 3/22 (14%) mothers and 0/22 (0%) infants had an antibody titer above the cut-off value (NCT ) AAV, adeno-associated virus; nc, not controlled. *If maternal AAV9 antibody titers were >1:50, the mother was asked to refrain from breastfeeding until 30 days post treatment. Day et al. Poster 11The 23rd International Annual Congress of the World Muscle Society, Mendoza, Argentina, October 2–6, 2018

QUESTIONS Please kindly submit your response to this question using the voting keypads provided Question 1: In your view, what would be the most effective strategy to reduce the impact of anti-AAV antibodies in gene therapy for neuromuscular disorders? 1 = Establishing anti-AAV antibody levels in patients prior to treatment 2 = Using an AAV capsid which was engineered to eliminate neutralizing antibody epitopes 3 = Clinical approaches such as plasmapheresis or circulating B cell reduction 4 = Being able to deliver the vector directly into a tissue or immune privileged site AAV, adeno-associated virus

QUESTIONS Please kindly submit your response to this question using the voting keypads provided Question 2: What is your main concern regarding the presence of immunity in patients treated with gene therapy for neuromuscular disorders? 1 = Reduction of treatment effectiveness 2 = Safety 3 = Potential limitation of use of gene therapy in patients with antibodies AAV, adeno-associated virus

CONCLUSIONS: CONSIDERATIONS FOR GENE THERAPY IN PRACTICE
AAV capsid binding and neutralizing antibodies can limit the effectiveness of gene therapy An effective strategy to prevent treatment failure is exclusion of patients with pre-existing anti-AAV antibodies Prevalence of anti-AAV antibodies is low against AAV serotypes adapted for gene therapy, particularly in young children Circulating maternal antibodies are observed in small numbers of infants; therefore there may be an optimal timing for administration of gene therapy Progress towards treating every patient will require innovation in AAV capsid design, a transient reduction in plasma cells, or reduction of the anti-AAV antibodies they produce AAV, adeno-associated virus

BIOSAFETY CONSIDERATIONS FOR THE CLINICAL USE OF AAVS
CHRIS JENKINS, PhD, MPH, RBP, CHMMMD Principal Partner & Chief Gene Therapy Biosafety Officer at Clinical Biosafety Services, USA This meeting is not part of the AAN Annual Meeting official programming and no CME is given for attendance. This Industry Therapeutic Update is sponsored and organized by AveXis.. © AveXis, Inc. All Rights Reserved MED-CON-UNB US 05/2019

DISCLOSURES Chris Jenkins, PhD, MPH, RBP, CHMMMD Founder of a for-profit clinical research organization supporting gene and cell therapy

SPEAKER PROFILE Principal Partner & Chief Gene Therapy Biosafety Officer of Clinical Biosafety Services Prior positions University of Texas at Austin WIRB-Copernicus Group (WCG) Biosafety University of Missouri The Scripps Research Institute Saint Louis University IBC Chair at 600+ convened IBC Meetings IBC, Iinstitutional Biosafety Committee

POINTS TO COVER Overview of biological risk in the context of gene therapies Explore institutional readiness for gene therapies Explain the risks and containment practices when working with AAV vectors AAV: adeno-associated virus

OVERVIEW OF BIOLOGICAL RISK IN CONTEXT OF GENE THERAPIES

OVERVIEW The risk of gene therapy agents is based on:
Risk of parental (wild type) delivery vector Nature of manufacturing and formulation Nature of genetic modifications Nature of administration (dosing) The appropriate containment for gene therapy agents is based on: Physical containment Work handling practices Resources for establishing risk and containment worldwide include: The NIH (NIH Guidelines)1 The CDC (Biosafety in Microbiological and Biomedical Laboratories)2 The World Health Organization (Laboratory Safety Manual)3 European Association of Hospital Pharmacists (Guidance on the Pharmacy Handling of Gene Medicines)4 CDC, Centre for Disease Control; NIH, National Institute of Health 1. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines) (April, 2016) retrieved from (Access date: March 2019), 2. Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition (October, 2018), retrieved from (Access date: March 2019), 3. World Heath Organization, Laboratory biosafety manual Third edition (2004), retrieved from (Access date: March 2019), 4. Vulto et al, European Association of Hospital Pharmacists (EAHP) Guidance on the Pharmacy Handling of Gene Medicines. Guidance on the Pharmacy Handling of Gene Medicines 2007; 13: 29-39 Reference for NIH Guidelines: Reference for CDC BMBL: Reference for WHO LSM: Reference for EAHP:

RISK GROUPS Group 1 Group 2 Group 3 Group 4
The NIH Guidelines and WHO categorize wildtype infectious agents into Risk Groups1,2: Group 1 Group 2 Group 3 Group 4 Agents that are not associated with disease in healthy adult humans Agents that are associated with human disease which are rarely serious and for which preventive or therapeutic interventions are often available Agents that are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available (high individual risk but low community risk) Agents that are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available (high individual risk and high community risk) Lowest risk Highest risk NIH, National Institute of Health; WHO, World Health Organization 1. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines) (April, 2016) retrieved from (Access date: March 2019), 2. Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition (October, 2018), retrieved from (Access date: March 2019). Reference for NIH Guidelines: Reference for CDC BMBL:

RISK GROUPS (CONT.) Appendix B of the NIH Guidelines provides an extensive list of infectious agents for each Risk Group The CDC BMBL provides information on high-risk pathogens but not low-risk infectious agents – they refer to the NIH Guidelines for recombinant agents The WHO LBM provides limited information on specific infectious agents The Public Health Agency of Canada has published Pathogen Safety Data Sheets (PSDS) that provide risk-group information on a wide variety of microorganisms NIH, National Institute of Health; BMBL, Biosafety in Microbiological and Biomedical Laboratories; CDC, Centre for Disease Control; LBM, Laboratory Biosafety Manual; WHO, World Health Organization Reference for NIH Guidelines: Reference for Public Health Agency of Canada PSDS:

BIOSAFETY LEVELS (BSLs)
Biosafety Levels established worldwide by WHO and CDC Ensures safe handling of biological agents Includes facilities, practices, and engineering controls Levels progress from the lowest risk biological agents to the highest risk biological agents Levels build on the precautions and containment equipment of the previous level plus any additional precautions needed for higher risk of biological agents BSL-3 BSL-2 BSL-1 BSL-4 High risk biological agents Low risk biological agents CDC, Centre of Disease Control; WHO, World Health Organization World Heath Organization, Laboratory biosafety manual Third edition (2004), retrieved from (Access date: March 2019) In addition to Standard Microbial Practices and Bloodborne Pathogen Standard Precautions, there is a more expansive set of standards established for handling microbiological organism that are tailored to the specific level of hazard. These are called Biosafety Levels, or BSLs 1. Biosafety levels are established worldwide by WHO and local authorities 2. They pertain to facilities, practices, and what are called engineering controls. A good example of an engineering control is a biological Safety Cabinet. 3. The Biosafety Levels progress from the lowest risk microbes at BSL-1 to the highest risk microbes at BSL-4. 4. Each level builds on the facilities, practices, and engineering controls of the previous level, plus additional precautions needed for the increased risk associated with the next level. Reference: Laboratory Biosafety Manual:

BIOSAFETY LEVELS 1 – 4 BSL-1 BSL-2 BSL-3 BSL-4 Microbes that are not known to cause disease in healthy adults Examples: E.coli, AAV Practices Standard microbiological practices Open bench or table permitted Laboratory personnel have specific training Laboratory supervised by scientist with appropriate training Standard lab practices for food, drink, smoking etc. apply Equipment Lab coat and gloves Facilities Sink for washing hands Means for controlling access (e.g. door) Microbes that pose moderate risk to workers and environment Example: Staphylococcus aureus Access to work area limited when work is conducted PPE includes mask and eye protection of face shield BSC for procedures that may cause exposure to aerosol or splashes Access to autoclave Work area includes self-closing doors and access to eye wash station Microbes that can cause serious or potentially lethal disease Example: Macrobacterium tuberculosis (tuberculosis) Receive immunization for microbes used Access restricted at all times BSC (preferably class II or III) for all open procedures Exhausted air cannot be recirculated Two sets of self-closing locked doors for entrance Immediate access to autoclave Hand washing sink near lab exit Method of decontaminating all lab wastes Very few labs in the world Most exotic and dangerous microbes (e.g. Ebola virus) Dedicated lab clothing Shower upon exit Class 3 BSC or full-body air supplied suit Separate building or isolated zone Dedicated air supply and processed exhaust AAV, adeno-associated virus; BSC, Biological Safety Cabinet; BSL, Biosafety Level; PPE, personal protective equipment Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition (October, 2018), retrieved from (Access date: March 2019). The lowest biosafety level is Biosafety Level 1, or BSL1. 1. BSL-1 is used for work with microbes that are not know to cause disease in humans. Examples include the bacteria E. coli found as part of the natural gut flora, as well as AAV9 2. The practices used at BSL-1 include: Standard Microbiological Practices Work on open benches or tables is permitted 3. Special equipment needed at BSL-1 include a lab coat and gloves. 4. Special facilities needed at BSL-1 include: A sink for washing hands, and A means of controlling access to the work area, such as a door Reference: Laboratory Biosafety Manual:

BIOSAFETY LEVELS 1 – 2 BSL-1 BSL-2
Microbes that are not known to cause disease in healthy adults Examples: E.coli, AAV Practices Standard microbiological practices Open bench or table permitted Laboratory personnel have specific training Laboratory supervised by scientist with appropriate training Standard lab practices for food, drink, smoking etc. apply Equipment Lab coat and gloves Facilities Sink for washing hands Means for controlling access (e.g. door) Microbes that pose moderate risk to workers and environment Example: Staphylococcus aureus Access to work area limited when work is conducted PPE includes mask and eye protection of face shield BSC for procedures that may cause exposure to aerosol or splashes Access to autoclave Work area includes self-closing doors and access to eye wash station AAV, adeno-associated virus; BSC, Biological Safety Cabinet; BSL, Biosafety Level; PPE, personal protective equipment Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition (October, 2018), retrieved from (Access date: March 2019). The lowest biosafety level is Biosafety Level 1, or BSL1. 1. BSL-1 is used for work with microbes that are not know to cause disease in humans. Examples include the bacteria E. coli found as part of the natural gut flora, as well as AAV9 2. The practices used at BSL-1 include: Standard Microbiological Practices Work on open benches or tables is permitted 3. Special equipment needed at BSL-1 include a lab coat and gloves. 4. Special facilities needed at BSL-1 include: A sink for washing hands, and A means of controlling access to the work area, such as a door Reference: Laboratory Biosafety Manual:

DECISION TREE FOR HANDLING OF GENE THERAPY PRODUCTS
Transgene is toxic or oncogenic Consider the risk group of the transgene Nonviable rDNA (plasmids, liposomes) Transgene is NOT toxic or oncogenic BSL-1 Transgene is toxic or oncogenic Consider the risk group of the organism and transgene Does NOT generally integrate into genome Gene therapy product Replication competent Transgene is NOT toxic or oncogenic Consider the risk group of the organism Integrates into genome Consider the risk group of the organism and transgene Viable bacteria, yeasts, viruses Transgene is toxic or oncogenic Consider the risk group of the transgene Does NOT generally integrate into genome Replication incompetent Transgene is NOT toxic or oncogenic BSL-1 Integrates into genome Consider the risk group of the organism and transgene BSL, Biosafety level, rDNA, recombinant DNA Petrich J, et al. Gene replacement therapy: A primer for the health-system pharmacist (manuscript in preparation). <<General walk through of AAV products>> Reference: Petrich J, et al. Gene replacement therapy: A primer for the health-system pharmacist (manuscript in preparation).

Transgene is toxic or oncogenic Consider the risk group of the transgene Nonviable rDNA (plasmids, liposomes) Transgene is NOT toxic or oncogenic BSL-1 Transgene is toxic or oncogenic Consider the risk group of the organism and transgene Does NOT generally integrate into genome Gene therapy product Replication competent Transgene is NOT toxic or oncogenic Consider the risk group of the organism Integrates into genome Consider the risk group of the organism and transgene Viable bacteria, yeasts, viruses Transgene is toxic or oncogenic Consider the risk group of the transgene Does NOT integrate into genome Does NOT generally integrate into genome Replication incompetent Transgene is NOT toxic or oncogenic BSL-1 Integrates into genome Consider the risk group of the organism and transgene BSL, Biosafety level, rDNA, recombinant DNA Petrich J, et al. Gene replacement therapy: A primer for the health-system pharmacist (manuscript in preparation). <<General walk through of AAV products>> Reference: Petrich J, et al. Gene replacement therapy: A primer for the health-system pharmacist (manuscript in preparation).

Viable bacteria, yeasts, viruses Transgene is toxic or oncogenic Consider the risk group of the transgene Does NOT generally integrate into genome Does NOT integrate into genome Replication incompetent Transgene is NOT toxic or oncogenic BSL-1 Integrates into genome Consider the risk group of the organism and transgene BSL, Biosafety level, rDNA, recombinant DNA Petrich J, et al. Gene replacement therapy: A primer for the health-system pharmacist (manuscript in preparation). <<General walk through of AAV products>> Reference: Petrich J, et al. Gene replacement therapy: A primer for the health-system pharmacist (manuscript in preparation).

PREPARATION OF INSTITUTIONS FOR AAV-GENE THERAPIES

BIOLOGICAL PROPERTIES OF AAV
AAV is a member of the parvovirus family of single-stranded small DNA viruses1 AAV requires a helper virus such as adenovirus or herpes simplex virus for replication1 AAV has several serotypes that impact tropism (susceptible tissues), but all appear to be non-pathogenic2 AAV-based vectors typically exist as extrachromosomal episomes1 AAV can efficiently infect both non-dividing and dividing cells1 AAV is typically transmitted by respiratory and gastrointestinal routes2,3 AAV, adeno-associated virus 1. Deyle DR, Russell DW. Curr Opin Mol Ther 2009;11(4): , 2. Concalves MAFV. Virol J 2005;2: ), 3. Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition (October, 2018), retrieved from (Access date: March 2019) References: Deyle DR, Russell DW Adeno-associated virus vector integration. Curr Opin Mol Ther 11(4): Concalves MAFV Adeno-associated virus: from defective virus to effective vector. Virol J 2:43

SAFETY FEATURES OF AAV VECTORS
Native (wild type) AAV is considered a Risk Group 1 microorganism – contact with AAV is not associated with disease in humans1 AAV vectors are replication-incompetent (non-infectious) by design1,2 Absent hazardous transgenes, AAV vectors can be handled at BSL-1 containment (BSL-2 containment may be considered for genes of unknown significance) AAV vectors are susceptible to common disinfectants approved for bloodborne pathogens (e.g. EPA Lists B, D, and E)3 AAV, adeno-associated virus; BSL, Biosafety level; EPA, Environmental Protection Agency 1. Conclaves MAFV. Virol J 2005;2: Deyle DR, Russell DW. Curr Opin Mol Ther 2009;11(4):442-44) EPA Disinfectant lists: References: EPA Disinfectant lists:

SAFETY CONSIDERATIONS OF AAV VECTORS
AAV vectors are biologically active and efficient at one-time gene transfer1 AAV vectors can be easily transmitted by aerosols2 – special care is needed to minimize and protect against aerosols Therapeutic genes present in AAV vectors may have varied properties3 Exposure to AAV vectors can result in seroconversion (false positive for exposure to native AAV) 3,4 ✘ AAV, adeno-associated virus 1. Foust KD, et al. Nat Biotechnol 2010;28(3):271–274, 2. Deyle DR, Russell DW. Curr Opin Mol Ther 2009;11(4): , 3. Tenenbaum et al, Evaluation of Risks Related to the Use of Adeno-Associated Virus-Based Vectors. Curr Gene Ther 2003; 3: , 4. Nayak S et al. Gene Ther 2011;17(3):

SAFETY CONSIDERATIONS OF AAV VECTORS: SHEDDING
Determining the AAV vector bio-distribution and shedding is central for the safety assessment of proposed early-phase clinical trials1 Diverse AAV vectors are shed by different routes and warrant different considerations and handling, based on the individual product in question1,2 Potential routes of shedding include tears, stool, saliva, urine and semen1,2 Shed AAV-based vectors are not expected to be infectious2 Product-specific studies should always inform handling instructions AAV, adeno-associated virus 1. Le Guiner C. et al. Methods Mol Biol. 2011;807: ), 2. Summary Notification Information Format for the Release of Genetically Modified Organisms Other Than Higher Plants In Accordance With Article 11 Of Directive (2002), retrieved from: (Access date: March 2019)

GUIDANCE ON HANDLING OF GENE THERAPIES
In North America, currently no formal guidance on handling of gene therapies exists General guidance was published by the University of Kentucky on handling of gene therapies Main aims include:1 Development of institutional readiness for gene therapies Standardizing practices of storage, transportation, preparation, dispensing, administration, waste disposal, decontamination and accidental exposure 1. Armitstead, JA, et al. Hospital Pharmacy. 2001;36(1):56–66. Armitstead, J. A., Zillich, A. J., Williams, K. L., Sitzlar, S. C., & Wermeling, D. (2001). Hospital and Pharmacy Departmental Policies and Procedures for Gene Therapy at a Teaching Institution. Hospital Pharmacy, 36(1), 56–66

SECTION ONE: SUMMARY 1 Hazardous drug handling mimics gene therapy Combined, an institution would be able to handle gene therapy effectively and safely. 2 Engineering Controls (Biological Safety Cabinet1,2 & USP 797*) 3 Work Practices and Training1,2 (equivalent to hazardous materials) 4 Personal Protective Equipment1 (standard gear for USP 797) 5 Risk assessment on each gene therapy agent (RG1 vs. RG2)1 *USP 797 is a far-reaching regulation that applies to health care institutions, pharmacies, physicians practice facilities, and other facilities in which compound sterile preparations are prepared, stored, and dispensed 1. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines) April 2. Fredrickson, Donald S. "Summary of the Proposed Guidelines for Research on Recombinant DNA Molecules." Draft. Summary. 27 February

BIOSAFETY CONSIDERATIONS FOR PHARMACY STAFF – STORAGE
Wear suitable disposable personal protective equipment when removing gene therapies from container in which it was delivered1 Store at a suitable temperature, according to product information1 Gene therapy storage areas should be labeled to alert employees of possible hazard1 The shipping container should be appropriately inspected by a pharmacist to ensure safe transit1 1. Armitstead, JA, et al. Hospital Pharmacy. 2001;36(1):56–66. Armitstead, J. A., Zillich, A. J., Williams, K. L., Sitzlar, S. C., & Wermeling, D. (2001). Hospital and Pharmacy Departmental Policies and Procedures for Gene Therapy at a Teaching Institution. Hospital Pharmacy, 36(1), 56–66

BIOSAFETY CONSIDERATIONS FOR PHARMACY STAFF – HANDLING & PREPARATION
Wear suitable protective clothing to minimize risk of microbiological contamination during preparation1 Use of a Class II biological safety cabinet or pharmaceutical grade isolator (compliant with NSF49 standard)1 Use appropriate personal protection equipment – coat and gloves1 Ensure decontamination of work surface areas with appropriate disinfectant and biohazard disposal1 Drug should be drawn up using ‘double-glove’ technique1 1. Armitstead, JA, et al. Hospital Pharmacy. 2001;36(1):56–66. Armitstead, J. A., Zillich, A. J., Williams, K. L., Sitzlar, S. C., & Wermeling, D. (2001). Hospital and Pharmacy Departmental Policies and Procedures for Gene Therapy at a Teaching Institution. Hospital Pharmacy, 36(1), 56–66

BIOSAFETY CONSIDERATIONS FOR PHARMACY STAFF – DISPENSING
All gene therapies should be prepared in a Class II hood1 Gene therapy prescription should be checked according to the normal pharmacy procedure1 Personal protective clothing standard for pharmacy should be worn as recommended1 The pharmacist must document dispensing specifics such as time, concentration volume and lot number1 All preparations must be double checked by another pharmacist1 1. Armitstead, JA, et al. Hospital Pharmacy. 2001;36(1):56–66. Armitstead, J. A., Zillich, A. J., Williams, K. L., Sitzlar, S. C., & Wermeling, D. (2001). Hospital and Pharmacy Departmental Policies and Procedures for Gene Therapy at a Teaching Institution. Hospital Pharmacy, 36(1), 56–66

BIOSAFETY CONSIDERATIONS FOR CLINICAL STAFF – ACCIDENTAL EXPOSURE
A spillage kit must be made available in the event of accidental exposure1 Areas must be immediately decontaminated following spillage1 Spillages must be contained by closing off the area in which the spillage has occurred1 Decontamination should be carried out according to local organizational guidelines1 Infection control should be notified of the spillage1 1. Armitstead, JA, et al. Hospital Pharmacy. 2001;36(1):56–66. Armitstead, J. A., Zillich, A. J., Williams, K. L., Sitzlar, S. C., & Wermeling, D. (2001). Hospital and Pharmacy Departmental Policies and Procedures for Gene Therapy at a Teaching Institution. Hospital Pharmacy, 36(1), 56–66

BIOSAFETY CONSIDERATIONS FOR CLINICAL AND PHARMACY STAFF – DISPOSAL
Disposable materials and personal protective equipment used in dispensing should be sealed in biohazard waste container and incinerated1 Any sharps used should be placed into a sharps container1 Spillage on clothes should be contained before leaving any spillage site, and any clothes should be discarded into a bag and then placed into an autoclave1 The waste container must be clearly labeled and display a biohazard symbol1 1. Armitstead, JA, et al. Hospital Pharmacy. 2001;36(1):56–66. Armitstead, J. A., Zillich, A. J., Williams, K. L., Sitzlar, S. C., & Wermeling, D. (2001). Hospital and Pharmacy Departmental Policies and Procedures for Gene Therapy at a Teaching Institution. Hospital Pharmacy, 36(1), 56–66

QUESTIONS Please kindly submit your response to this question using the voting keypads provided Question 1: What level of experience does your institution have in handling modified AAV vectors? 1 = Extensive experience 2 = Some experience 3 = Limited experience 4 = No experience

QUESTIONS Please kindly submit your response to this question using the voting keypads provided Question 2: How would you describe your institution’s level of readiness for the handling of modified AAV vectors? 1 = Very high 2 = High 3 = Moderate 4 = Low 5 = Very low

CONCLUSIONS Guidance on handling of gene therapies has been set out by NIH/CDC1,2 The biological risks from working with AAV vectors are considered very low AAV vectors can be handled at BSL-1, the lowest Biosafety level Formal staff training is imperative for the safe and effective handling of gene therapies Institutional readiness will enable quick and safe uptake of novel gene therapies AAV, adeno-associated virus; BSL, Biosafety level; CDC, Centre for Disease Control; NIH; National Institute of Health 1. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines) (April, 2016) retrieved from (Access date: March 2019), 2. Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition (October, 2018), retrieved from (Access date: March 2019).

PANEL DISCUSSION This meeting is not part of the AAN Annual Meeting official programming and no CME is given for attendance. This Industry Therapeutic Update is sponsored and organized by AveXis. © AveXis, Inc. All Rights Reserved MED-CON-UNB US 05/2019

EVALUATION QUESTIONS This meeting is not part of the AAN Annual Meeting official programming and no CME is given for attendance. This Industry Therapeutic Update is sponsored and organized by AveXis. © AveXis, Inc. All Rights Reserved MED-CON-UNB US 05/2019

EVALUATION QUESTIONS Please kindly submit your response to this question using the voting keypads provided Question 1: Did the information presented today provided you with new insights into the use of gene therapies in the treatment of neuromuscular disorders? 1 = Strongly agree 2 = Agree 3 = Neither agree nor disagree 4 = Disagree 5 = Strongly disagree

EVALUATION QUESTIONS Please kindly submit your response to this question using the voting keypads provided Question 2: After this session, how would you describe your current level of knowledge on gene therapy and its use in treating neuromuscular disorders? 1 = Very high 2 = High 3 = Moderate 4 = Low 5 = Very low

EVALUATION QUESTIONS Please kindly submit your response to this question using the voting keypads provided Question 3: Was the content of this Industry Therapeutic Update informative and useful to your work? 1 = Very high 2 = High 3 = Moderate 4 = Low 5 = Very low

Associate Professor of Neurology,
CLOSING REMARKS Perry Shieh, MD, PhD Associate Professor of Neurology, University of California, Los Angeles, USA This meeting is not part of the AAN Annual Meeting official programming and no CME is given for attendance. This Industry Therapeutic Update is sponsored and organized by AveXis. © AveXis, Inc. All Rights Reserved MED-CON-UNB US 05/2019

INDUSTRY THERAPEUTIC UPDATE OBJECTIVES
Explore the progress and current clinical landscape of gene therapies Understand the fundamentals of gene therapy including the potential application of gene therapy for neuromuscular disorders Discuss the translation of gene therapy into clinical practice, including the safe handling of vectors

THANK YOU This meeting is not part of the AAN Annual Meeting official programming and no CME is given for attendance. This Industry Therapeutic Update is sponsored and organized by AveXis. © AveXis, Inc. All Rights Reserved MED-CON-UNB US 05/2019

WELCOME AND INTRODUCTION Perry Shieh, MD, PhD

Similar presentations

Presentation on theme: "WELCOME AND INTRODUCTION Perry Shieh, MD, PhD"— Presentation transcript:

Similar presentations

About project

Feedback

Log in

Auth with social network:

WELCOME AND INTRODUCTION Perry Shieh, MD, PhD

Similar presentations

Presentation on theme: "WELCOME AND INTRODUCTION Perry Shieh, MD, PhD"— Presentation transcript:

Similar presentations

About project

Feedback