Assessing the Risks of Viral Vector Protocols

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InstitutionalInstitutional BiosafetyBiosafety Committees:Committees: PromotingPromoting OptimalOptimal PracticePractice NowNow andand inin thethe FutureFuture

AssessingAssessing thethe RisksRisks ofof ViralViral VectorVector ProtocolsProtocols

BruceBruce Crise,Crise, PhD,PhD, CBSPCBSP

Advanced Technology Program OutlineOutline z ViralViral VectorVector ProtocolProtocol Review:Review: – What to look for – How to make it safe z RoleRole ofof thethe IBCIBC – NIH Guidelines for Research Involving Recombinant DNA z HowHow toto assessassess riskrisk – The registration form – What the IBC needs to know – The underlying biology of the virus/vector system and transgene being expressed – Quality assurance requirements OutlineOutline ContinuedContinued z MitigationMitigation ofof risksrisks – Process controls – Training & Testing

Fluorescent markers and their application – Engineering controls – PPE – Vaccination – Quality control

Virology and Molecular Biology tool kit z Animals:Animals: AddedAdded RisksRisks RoleRole ofof thethe IBCIBC z ToTo obtainobtain aa fullfull understandingunderstanding ofof thethe riskrisk associatedassociated withwith viralviral vectorvector researchresearch z ToTo provideprovide aa comprehensivecomprehensive reviewreview andand independentindependent riskrisk assessmentassessment ofof thethe researchresearch

z Subject matter experts z ToTo ensureensure thatthat experimentsexperiments areare conductedconducted safely,safely, andand thatthat appropriateappropriate measuresmeasures areare usedused toto mitigatemitigate risksrisks NIHNIH Guidelines:Guidelines: RiskRisk GroupsGroups

Risk Group 1 (RG1) Agents that are not associated with disease in health adult humans Risk Group 2 (RG2) Agents that are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available Risk Group 3 (RG3) 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) Risk Group 4 (RG4) 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) AppendixAppendix BB--I.I. RiskRisk GroupGroup 11 (RG1)(RG1) AgentsAgents

RG1 agents are not associated with disease in healthy adult humans. ExamplesAdeno-associated of RG1 agents include virus… adeno -(AAV)associated types virus (AAV) 1-4 types 1 through 4; and recombinant AAV constructs, in which the transgeneRecombinant does not encode AAV either with a potentially benign tumorigenic transgene gene product or a toxin molecule and are produced in the absence of a helperand virus without… helper virus contamination Those agents not listed in Risk Groups (RGs) 2, 3 and 4 are not automaticallyMuLV and or implicitly MuLV classified recombinants in RG1; a risk assessmentencodingmust be conducted based on the known and potential properties of the agentsbenign and their transgenes relationship to agents that are listed. RiskRisk GroupsGroups DescribedDescribed inin NIHNIH GuidelinesGuidelines

Adenoviruses, human - all types Paramyxoviruses Alphaviruses (Togaviruses) - Group A Arboviruses –Newcastle disease virus –Eastern equineAdenoviruses, encephalomyelitis virus human—all–Measles virus types –Venezuelan equine encephalomyelitis vaccine –Mumps virus strain TC-83 –Parainfluenza viruses types 1, 2, 3, and 4 –Western equine encephalomyelitis virus –Respiratory syncytial virus CoronavirusesHerpesviruses—except Herpesvirus simiae Parvoviruses Flaviviruses (Togaviruses) - Group B Arboviruses –Human parvovirus (B19) –Dengue virus serotypes 1, 2, 3, and 4 Picornaviruses –Yellow fever virus vaccine strain 17D Poxviruses, except restricted–Polioviruses - all types,groups wild and attenuated Hepatitis A, B, C, D, and E viruses –Rhinoviruses - all types Herpesviruses - except Herpesvirus simiae (Monkey B virus) Poxviruses - all types except Monkeypox virus and restricted poxviruses including Alastrim, –Cytomegalovirus Smallpox, and Whitepox –Epstein Barr virus Reoviruses - all types including –Herpes simplex types 1 and 2 Rhabdoviruses –Herpes zoster –Rabies virus - all strains –Human herpesvirus types 6 and 7 –Vesicular stomatitis virus – laboratory adapted strains including VSV-Indiana, San Juan, and Orthomyxoviruses Glasgow –Influenza viruses types A, B, and C Togaviruses (see Alphaviruses and Flaviviruses) Papovaviruses –Rubivirus (rubella) –All human papilloma viruses RiskRisk GroupsGroups DescribedDescribed inin NIHNIH GuidelinesGuidelines

Alphaviruses (Togaviruses) - Group A Arboviruses –Semliki Forest virus –Venezuelan equine encephalomyelitis virus (except the vaccine strain TC-83, see Appendix B-II-D (RG2)) Flaviviruses (Togaviruses)Retroviruses - Group B Arboviruses –Japanese encephalitis•HIV-1 virus and HIV-2 –Yellow fever virus Poxviruses • HTLV-1 and HTLV-2 –Monkeypox•SIV virus Retroviruses –Human immunodeficiency virus (HIV) types 1 and 2 –Human T cell lymphotropic virus (HTLV) types 1 and 2 –Simian immunodeficiency virus (SIV) Rhabdoviruses –Vesicular stomatitis virus RiskRisk GroupsGroups DescribedDescribed inin NIHNIH GuidelinesGuidelines

Arenaviruses –Guanarito virus –Lassa virus –Junin virus –Machupo virus –Sabia Bunyaviruses (Nairovirus) –Crimean-Congo hemorrhagic fever virus Filoviruses –Ebola virus –Marburg virus Flaviruses (Togaviruses) - Group B Arboviruses –Tick-borne encephalitis virus Herpesviruses (alpha) –Herpesvirus simiae (Herpes B or Monkey B virus) Paramyxoviruses –Equine morbillivirus Hemorrhagic fever agents and viruses as yet undefined TheThe ReviewReview ShouldShould FollowFollow BiologyBiology andand CommonCommon SenseSense

Basic Virus Specific Biosafety Biosafety

As safe as reasonably possible IBCIBC FormsForms andand ResponsesResponses

Calvin tries filling out an IBC form…

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…and working on the IBC

Cartoon Image Removed IBCIBC FormsForms andand ResponsesResponses z TheThe formform shouldshould askask thethe rightright questionsquestions z TheThe formform shouldshould provideprovide somesome guidance,guidance, butbut notnot specificspecific answersanswers z WhatWhat shouldshould thethe IBCIBC dodo whenwhen thethe PIPI doesndoesn’’tt understandunderstand thethe problem?problem? z WhatWhat aboutabout reagents/animalsreagents/animals generatedgenerated elsewhereelsewhere (commercial(commercial andand noncommercialnoncommercial sources)?sources)? CustomCustom producedproduced viralviral vectorvector stocksstocks andand ““homehome growngrown”” vectorvector systemssystems – Quality Control Issues WhatWhat thethe IBCIBC NeedsNeeds toto KnowKnow z What viral vectors will be used? z What experiments will be done with recombinant DNA and/or viral vectors? z Will anything be done that would extend the host range or enhance the pathogenicity of the vectors? z Is it reasonable to expect that the vectors can be complimented or recombine in the proposed experiments? z What will be done to minimize the risks in the proposed experiments? z Reagent cycle: “Cradle to grave” IBCIBC FormsForms andand ResponsesResponses z HowHow toto handlehandle ““blanketblanket”” protocolsprotocols thatthat covercover manymany kindskinds ofof vectorvector systemssystems andand aa myriadmyriad ofof expressedexpressed genesgenes z WhatWhat wonwon’’tt bebe donedone inin thethe experimentsexperiments cancan bebe asas importantimportant asas whatwhat willwill bebe donedone BasicBasic BiosafetyBiosafety ConcernsConcerns z What is the host range of the parental virus? (infection vs. replication) z Has anything been done to extend the host range of the vector? z What is the pathogenicity of the parental virus? z Has anything been done to extend the pathogenicity? (oncogenes, toxin genes, etc.) z Can the recombinant DNA be mobilized? (viral vs. nonviral DNAs…complementation vs. recombination) z Is the free DNA/RNA infectious? VirusVirus SpecificSpecific BiosafetyBiosafety ConcernsConcerns z What is the normal route of infection? (aerosols vs. direct contact) z Can the vector interact with endogenous viruses? (MLV vectors in murine cells) z Can the vector interact with exogenous viruses (human adenoviruses with adenovirus and AAV vectors) z If the vector is intended to be defective are there any replication competent recombinants in the stock? z Does the disinfectant/procedure inactivate the vector that is being used? RecombinantRecombinant DNADNA andand ViralViral VectorsVectors z WhatWhat isis aa virus?virus? – It’s small and hard to manipulate… z ViralViral lifelife cyclescycles andand viralviral lifelife stylesstyles z RecombinantRecombinant DNADNA applicationsapplications thatthat involveinvolve viralviral vectors:vectors: – Replication competent viral vectors – Replication defective viral vectors – Cells and animals with viral vectors – Expression of genes (cDNAs, miRNA, etc) ThingsThings VirusesViruses DoDo…… DifferentDifferent viralviral vectorsvectors dodo thingsthings differentlydifferently z Modify host genome z Modify host immune response z Remain latent z Circulate in blood or remain in tissues z Shed from the host – Bedding and excreta – Aerosol z Pathogenic to host z Recombine with other viruses (can happen during production or in vivo after introduction into the animal) DevelopingDeveloping aa BroaderBroader SenseSense ofof RiskRisk forfor rDNArDNA andand ViralViral VectorsVectors z Biological function of transgene

z Biological control – Permissive host (or permissive grafted host) – Immunity for viral vector

z Immunity evoked by transgene – Human genes – Regulatory RNAs

e s t e s r e o s , n a o s n n a s a e e i e e A l s t s m g s i n u N : a A i o l d e t R s d n a m N g f h n h c i h s t o o s i Higher Lower m t i t e C hR i e c , i a s t U s n w n t s r w r c Risk Risk o L m e o s r d d i o , d g o p n e n n s a n t a e n t a o s r g g t i a a g e d e - s a i m e i r e k β c a r e c m u t p t r , o o n o u H n p c a P s F e s a e F H s u r M g s a s i G a d

Comprehensive gene information: http://atlasgeneticsoncology.org/index.html AsAs SafeSafe asas ReasonablyReasonably PossiblePossible

z Biological barriers are your best protection: If the vector won’t replicate in a human… z Physical barriers (BSCs, gloves, masks, clothing, etc.) are important, but they need to match the route of infection z Watch out for sharps/needles! z Your immune system is the final level of protection; try not to use it. (vaccination can help in some cases) z Know what you are working with: Quality control for cells, animals and vectors QualityQuality AssuranceAssurance z IsIs thethe laboratorylaboratory infrastructureinfrastructure capablecapable ofof supportingsupporting thethe experimentsexperiments z AreAre thethe SOPsSOPs suppliedsupplied withwith thethe IBCIBC registrationregistration sufficientsufficient toto covercover thethe activitiesactivities z HowHow currentcurrent areare thethe SOPsSOPs relativerelative toto thethe laboratorylaboratory activitiesactivities – IBC renewals and updates z MonitoringMonitoring laboratorylaboratory activitiesactivities WhatWhat’’ss BeingBeing DoneDone withwith thethe Vector?Vector? z TypesTypes ofof manipulationsmanipulations – Culturing, titering, concentration, purification z IntroductionIntroduction intointo animalsanimals – And will the viral vector be coming back out?

Cells and explants

Pathology samples

Wastes z DoesDoes thethe nucleicnucleic acidacid ofof thethe viralviral vectorvector presentpresent aa riskrisk LentiviralLentiviral VectorVector ConsiderationsConsiderations

Wild Type HIV-1 Particles Mutant HIV-1 Particles UsefulUseful GuidanceGuidance forfor LentiviralLentiviral VectorsVectors

http://oba.od.nih.gov/rdna_rac/rac_guidance_lentivirus.html z The number of recombination events needed to reassemble the virus z Is the entire virus present ? – env (and tat) deletions common in lentiviral vectors LentiviralLentiviral VectorsVectors z Env-deleted lentiviral vectors complimented by VSV- G do not appear to give rise to replicating viruses z Lentiviral vectors do not successfully recombine with any known endogenous viruses z Nature of transgene z Integration/insertional mutagenesis – One of the few instances where antivirals can block – But there has to be advnaced planning z Lentiviral vector infection of human cells can pose special risks z In some cases, the literature that comes with commercial lentiviral vectors is misleading z It is not easy to characterize a complex retroviral library (commercial or noncommercial) RecombinationRecombination z AreAre allall thethe sequencessequences neededneeded toto reconstitutereconstitute thethe virusvirus everever presentpresent inin oneone cell?cell? z SequenceSequence homologyhomology enhancesenhances thethe raterate ofof recombinationrecombination butbut recombinationrecombination stillstill happenshappens inin thethe absenceabsence ofof homologyhomology z RareRare eventsevents happenhappen frequentlyfrequently inin highhigh titertiter viralviral stocksstocks z ItIt onlyonly takestakes oneone replicationreplication competentcompetent recombinantrecombinant virusvirus…… MitigationMitigation MeasuresMeasures

Environment Behavior

Organism Vaccination

Cell Antivirals Antiodies

Inactivation methods virus ProcessProcess ControlsControls andand Training:Training: FluorescentFluorescent MarkersMarkers

z Fluorescent materials for tracking materials prior to use with agent/vector z Easily tracked with UV light – Illumination from a UV light in safety cabinet/hood – Hand-held UV light z Markers: – Riboflavin

200mg/L – Fluorescein

350mg/L ProcessProcess ControlsControls && TrainingTraining RiboflavinRiboflavin andand FluoresceinFluorescein

n avi ofl rib 5.0ul 1.0ul 0.1ul

fluorescein

Fluorescein photobleaches after exposure to ultraviolet light MaterialMaterial RetainedRetained onon ThreadsThreads ofof CapCap

Transfer materials to a clean tube to eliminate contamination PostPost--InjectionInjection LeakageLeakage WhereWhere’’ss thethe Spill?Spill? EngineeringEngineering ControlsControls andand PPEPPE z SharpsSharps !! z DoDo thethe physicalphysical barriersbarriers matchmatch thethe risk?risk? z LargeLarge scalescale considerationsconsiderations z IsIs thethe PPEPPE sufficientsufficient – Aerosols – Centrifugation z WorstWorst casecase scenariosscenarios – Spill drill SharpsSharps z InIn mostmost cases,cases, alternativesalternatives methodsmethods areare availableavailable

Nissin et al., (2003) Emerging Infectious Diseases, Volume 9, Number 6, June 2003. Accidental Infection of Laboratory Worker with Vaccinia MitigationMitigation ofof RiskRisk z EngineeringEngineering ControlsControls MitigationMitigation ofof RiskRisk z EngineeringEngineering ControlsControls contcont’’dd – Needle pointed away from hands PlanningPlanning forfor thethe WorstWorst

z WouldWould practicepractice withwith aa fluorescentfluorescent markermarker help?help?

1 liter of recombinant HSV vector z SpillSpill drills/spilldrills/spill cleanclean--upup kitskits z ContingencyContingency PlanningPlanning WouldWould VaccinationVaccination Help?Help?

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QualityQuality Control:Control: AreAre YouYou SureSure YouYou KnowKnow WhatWhat YouYou AreAre Getting?Getting? VirologyVirology ToolTool KitKit z DetectingDetecting andand validatingvalidating viruses/vectorsviruses/vectors – Cytopathic effects – Transformation – Molecular assays (ELISA, antibodies, PCR etc.) – When in doubt sequence the vector UsefulUseful WaysWays toto MonitorMonitor forfor ViralViral VectorVector QualityQuality z PCR/SequencePCR/Sequence z Plaque/ReplicationPlaque/Replication AssaysAssays z WhatWhat toto monitormonitor – Viral vector stocks – Producer cells – Transduced/carrier cells z WhatWhat toto monitormonitor for:for: – RCA – Endogenous contaminants ExperimentsExperiments InvolvingInvolving AnimalsAnimals z InteractionInteraction betweenbetween thethe IBCIBC andand ACUCACUC committeescommittees isis neededneeded forfor comprehensivecomprehensive safetysafety programprogram z CoCo--mingledmingled membershipsmemberships betweenbetween thethe twotwo committeescommittees leadsleads toto consistencyconsistency inin thethe reviewreview processprocess z IBCIBC andand ACUCACUC formsforms withwith complementarycomplementary questionsquestions resultsresults inin thoroughthorough reviewreview AcknowledgementsAcknowledgements z SteveSteve HughesHughes z AlanAlan KaneKane

z JonathanJonathan SummersSummers z JoeJoe KozlovacKozlovac

z JulieJulie BullockBullock

z MistyMisty HawesHawes

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[email protected]@mail.nih.gov