Emerging Laboratory Safety and Health Issues

Emerging Laboratory Safety and Health Issues Diane Liu, MD, MPH Chief, Occupational Health (formerly) UC Berkeley Disclosure I have no financial conflicts. Off label use of medications will be discussed UC System Occupational Health 10 campuses Includes medical centers and campuses Office of the President UC Agriculture and Natural Resources http://ucanr.edu/about_anr/we_are_uc_anr/ 7 on site Occupational Health Clinics Campus workers compensation treatment Laboratory occupational health Institutional Biosafety Committee Animal Care and Use Committee Campus/MC Occupational Health and Wellness 1

University of California Berkeley Title FTE Faculty Ladder rank and Equivalent 1367.7 Faculty Clinical/In Residence/Adjunct 141.7 Faculty Lecturers 406.5 Other Academic Employees 962.2 Post doctoral Scholars 877.1 Student Teaching/Research Assistants 1792.4 Senior Management 10.5 MSP Managers 517.8 MSP Senior Professionals 790.2 PSS Non Students 6314.3 Student Staff 959.9 FTE Academic 5484.7 Non Academic 8592.7 Total 14077.3 2

University of California Berkeley Title Headcount Faculty Ladder rank and Equivalent 1513 Faculty Clinical/In Residence/Adjunct 214 Faculty Lecturers 797 Other Academic Employees 1348 Post doctoral Scholars 1208 Student Teaching/Research Assistants 4722 Senior Management 13 MSP Managers 518 MSP Senior Professionals 863 PSS Non Students 7104 Student Staff 5257 Headcount Academic 9802 Non Academic 13755 Total 23557 Reporting of Laboratory Accidents National Institutes of Health Office of Science Policy Office of Laboratory Animal Welfare OSHA Laboratory Accidents UCB 2016 17 Lab Accidents identified: 16 Total injuries: 500 600/yr Injury Cut 3 Needlestick 2 Skin exp, with PPE 2 Skin exp, no PPE 1 Splash with PPE 2 Splash no PPE 5 Resp irritant 1 3

In the news Boston College Student Hurt in Lab Accident Boston Globe 2011 Colorado College Lab Accident Hospitalizes 13 The Denver Post 2013 Death in the Lab Discover 2015 Dartmouth Faces Fine of $13,000 Valley News 1997 Research at Berkeley World Class Research 8 current Nobel Laureates 144 members of National Academy of Sciences 235 Fellow of the American Academy of Arts and Sciences Innovative and Entrepreneurial Currently there are ~450 operating laboratories UC Berkeley Faculty Health Programs Occupational Health Clinic Campus Employee Health Resource for Health in Research Laboratories MD 2.2 FTE NP 1.0 FTE AA 2.0 FTE MA 3.0 FTE Wellness Coordinator 1.0 FTE Registered Dietician 0.2 FTE 4

Rabies Ancient Disease Modern times A condition known for 4000 years Democritis in 4 th Century BC, gave a clear description of rabies Aristotle (300 BC) notes that rabies as disease of dogs and any animal the dog bites Rabies: Latin to rage (rabere) 16 th century, Girolamo Fracastoro discovered that rabies was a fatal disease affecting humans as well as animals, calling it "an incurable wound. Louis Pasteur a French biologist that created the first vaccine in 1885. Etymologia: Rabies. Emerg Infect Dis. 2012;18(7):1169. https://dx.doi.org/10.3201/eid1807.et1807 Ancient Disease Modern times Canines new WHO initiative (Jan 2018) to eradicate rabies (a neglected zoonotic disease) in developing countries Wild animals: carnivores, raccoons, skunks, foxes No rabies seen in Antarctica Current incidence in the U.S. is less than 2 per year ( 100 per year in the late 1800s to early 1900s) Canine vaccination very successful in the reduction of rabies in developing countries. 5

By Wei Chung Allen Lee, Hayden Huang, Guoping Feng, Joshua R. Sanes, Emery N. Brown, Peter T. So, Elly Nedivi Neuroscience Imagine being able to choose one neuron and identify all of the neurons connected to that single neuron. This is felt to be key to fully understand the functioning of the brain Scientists at the Salk Institute reported successfully turning the deadly rabies virus into a tool for neuroscience. Monosynaptic Restriction of Transsynaptic Tracing from Single, Genetically Targeted Neurons Wickersham, Ian R. et al. Neuron, Volume 53, Issue 5, 639 647. March 2007. Modified Rabies Virus (RV) Single gene deletion rabies Glycoprotein (G) This glycoprotein allows RV to move transynaptically from neuron to neuron from bite to the brain and then anterograde to the salivary glands. It is not involved in replication of the virus. If one deletes this gene, the rabies virus is marooned in the cell that has been infected. If you add the gene (G) via plasmid into the same cell, it will replicate and be able to transfect any synaptically connected neurons However, the modified virus cannot spread further: monosynaptic 6

Pseudo typing A second modification In order to further customize the virus, the investigators pseudotyped the virus with an avian envelope protein (EnvA) to only recognize a specific receptor, (TLV) Changes the tropism of the virus. No longer recognized mammalian cells, but recognize avian cells Can transfect specific neurons with plasmids with TLV gene, G gene and a fluorophore such as Green Fluorescent Protein (EGF), mcherry or DsRed2. SAD G EGFP/EnvA is an example of a modified rabies virus. This modified version of the rabies virus forces neurons it infects to produce a green fluorescent protein. Occupational Health Issues 2008 First researcher at UCB requested to use this modified rabies virus. Only one gene different than wild type RV Presented to Institutional Biosafety Committee, determined with input from the NIH that all researchers and persons potentially exposed to virus have pre exposure prophylaxis for rabies infection. Mortality of RV infection approaches 100% in unvaccinated individuals. Vaccination offers close to 100% protection Latency: an eclipse phase for days to months Occupational Health Issues G deleted RV Current recommendations are to have pre exposure counselling for possibility of vaccination. Post exposure: follow current guidelines appropriate for prevaccinated vs. unvaccinated individuals 7

Sample SOP for G deleted rabies BL 2 containment: disposable gloves, gown and faceshield. Work to be done in dedicated BL 2 room All persons to be trained. Signage on doors Reduce level of sharps use. Proper sharps disposal Red biohazard bags for waste 10% bleach for decontamination of surfaces Animals injected with g deleted RV remain housed in BL 2 cages but can be moved to BL 1 rooms for housing. "Stanek E, Cheng S, Takatoh J, Han B, Wang F (2014).Monosynaptic premotor circuit tracing reveals neural substrates for oro motor coordination". elife. DOI:10.7554/eLife.02511. PMID 24843003. PMC: 4041139 Bats and Rabies Photos by Christie Ferrecchia, DVM 8

Research at UCB Field researchers Museum of Vertebrate Zoology specimen collection Live trapping Specimens from local health department Teaching classes in basic field technique Studying bat viruses and spillover events in Madagascar new! Rabies Vaccination pre exposure Recommended for: Veterinarians Persons who work with rabies virus Frequent contact with animals known to be infected with rabies Study bats, work in caves Travel to countries with canine rabies Pre exposure prophylaxis IM vaccine at 0, 7, 21 28 days No need for post vx titer Titer check every 1 or 2 years Avoid vx in immune compromised Rabies Post Exposure Vaccine Decontamination of wound: thorough gentle cleansing with soap and H 2 O. Povidone Iodine if available. If pre vaccinated Dose 1 as soon as possible after decontamination of wound Dose 2 3 days after first dose If not pre vaccinated Dose 1 as soon as possible after decontamination of wound. HRIG, infiltrated around wound and the rest IM distant from vx site Dose 2 3 days after first dose Dose 3 7 days after first dose Dose 4 14 days after first dose Dose 5 (for immune compromised host) 4 weeks after first dose 9

Viral Vectors Biosafety Considerations for Research with Lentiviral Vectors https://osp.od.nih.gov/wp content/uploads/2014/01/lenti_containment_guidance_0.pdf Viral Vectors Engineered viruses that take advantage of the infective and replicative qualities of the virus Introduce genetic material into the genome of cells, either in vitro or in vivo Risks include accidental exposure to laboratory workers to infectious viruses By biology pop (biology pop) [CC BY SA 4.0 (https://creativecommons.org/licenses/by sa/4.0)], via Wikimedia Commons Non retroviral vectors Some vectors used to introduce genes into host cells, such as adenovirus or adeno associated virus. Gene expression is typically transient because genes are not integrated into host genome, remain as plasmid Risks: may induce an immune response to viral proteins Risk seen in clinical trials Risk in research is theoretical. Adverse events have been documented in clinical trials of gene therapy No risk if bitten by an animal transfected with viral vector 10

Lentiviral Vectors HIV is a lentivirus in the genus Retroviridae HIV, as a viral vector inserts genetic material into host genome Can infect dividing and non dividing cells Wild type HIV is a safety issue for workers, so the virus has been engineered to achieve safe gene transfer HIV typically recognizes CD4 receptors, so has affinity for T cells, macrophages, microglial cells. This is limiting in research. To increase tropism, the envelope protein is often replaced with vesicular stomatitis virus glycoprotein (VSV G). Human Immunodeficiency Virus Wild type rev rev vpr gag vif tat vpu tat nef Ψ LTR L pol env LTR L gag core proteins and matrix proteins pol reverse transcriptase, integrase, protease rev regulator of viral expression tat regulation of transciption env transmembrane viral proteins (recognize CD4) Accessory genes: vif, vpr, vpu, nef LTR Long Terminal Repeats Ψ packaging signal First generation lentiviral vector promotor env rev rev promotor gag pol vif vpr tat vpu tat nef LTR L 11

Second generation lentivirus promotor env rev rev gag promotor pol tat tat LTR L promotor Transfer plasmid LTR L promotor env gag promotor pol rev LTF LTR promotor Transfer plasmid LTR L Third generation genes necessary for expression on three plasmids Packaging a 3 rd gen lentivirus 12

Safety of Lentivirus 3 rd gen Non replicative Requires several recombination events to revert to wild type HIV in the presence of HIV 4 th generation Creation of Self Inactivating (SIN) LVV Risks of Lentivirus and Transgene exposure Recombination and reversion to wild type HIV Oncogenic potential when using LVV Activation of oncogenes or inactivation of tumor suppressor genes Insertional mutagenesis Gene transduction of an oncogenic transgene LVVs might target multiple genes which could silence or inactivate tumor suppressor activity Unknown functions of some transgenes may promote oncogenesis or other toxic effects Transduction of a toxin gene Clinical gene therapy Howe SJ, Mansour MR, Schwarzwaelder K, et al. Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID X1 patients. The Journal of Clinical Investigation. 2008;118(9):3143 3150. doi:10.1172/jci35798. Hacein Bey Abina S, Garrigue A, Wang GP, et al. Insertional oncogenesis in 4 patients after retrovirus mediated gene therapy of SCID X1. The Journal of Clinical Investigation. 2008;118(9):3132 3142. doi:10.1172/jci35700. 13

Proposed Post exposure protocol Discussed at recent professional conferences: OBA, Eagleson Institute Journal of Occupational and Environmental Medicine Schlimgen R, Howard J, Wooley D, et al. Risks Associated With Lentiviral Vector Exposures and Prevention Strategies. Journal of Occupational and Environmental Medicine. 2016;58(12):1159 1166. Off label use of FDA approved medications Re framing treatment for researchers who have been convinced that use of late generation LVVs are safer Pre Exposure preparations Proposed recommendations Review biological hazard protocols for lentiviral exposure Consider nature of transgene Does it silence a tumor suppressor gene? Does it express an oncogene? A toxin? Expanded host range (pseudotyped?) Large volume? Create a post exposure protocol for each lab Train each lab member on the procedures for decontamination, notification and the need for immediate care. Use of medical cards for providers Post exposure first aid Call 911 if needed for injury requiring emergency treatment Begin decontamination: Intact Skin Exposure: immediately wash area with copious amounts of running water to dilute, cleanse and flush Nonintact Skin Exposure: immediately wash area with generous amounts soap and water to dilute, cleanse and flush Mucous Membrane Exposure: immediately flush the area with running water for at least 15 minutes Droplet exposure: see above depending on area of exposed Contact the institution s Biosafety Officer 14

Post exposure prophylaxis Integrase inhibitor plus nucleoside reverse transcriptase inhibitor Initiate ASAP, within 2 hours, but no later than 72 hours (optimally) 7 day course Non FDA approved A suggested regimen Raltegravir (Isentress) 400 mg po BID tenofovir (Truvada) 300 mg po once daily (Descovy may be used instead) Follow up for medication side effects Any sequelae from exposure may have long latency, and truly unknown effects. Integrase Inhibitor NRTI By Dan Cojocari [CC BY SA 3.0 (https://creativecommons.org/licenses/by sa/3.0) via Wikimedia Commons (adapted) PEP Considerations for LVV exposure Off label use of medications However safety profile of meds fairly well known Recommendations based on HIV PEP Risk of insertional mutagenesis or oncogene promotion is not quantifiable Integration thought to occur in 1 2 hours. PEP in time? Exposure registry? 15

Other antiviral PEP protocols HIV exposure PEP Isentress and Descovy for 28 days B virus (macacine herpes B virus 1) Valcyclovir or Acyclovir for 28 days CRISPR/Cas9 CRISPR= Clustered Repeating Interspaced Short Palindromic Repeats 16

What is CRISPR/Cas9 First identified in E. coli An immune system for bacteria against viruses Palindromic DNA interspaced with: Spacer DNA Match up with viral DNA When a virus attempts to infect the bacteria, a copy of the spacer DNA is created Cas proteins are also made and form into a complex Function is to unwind DNA( helicase) and cut DNA (nuclease) using the spacer genes to identify the viral genes What is CRISPR/Cas9 The modification done by researchers at UCB and the Broad Institute used the function of this bacterial system to create a tool to cut dsdna in very precise places Cells naturally detect and repair cuts in dsdna Can be used to either delete a gene (correct a mutation or knockout the gene) or insert new DNA (gene therapy) Cells are transfected with Cas9 genes and grna Can be done with many methods including using lentiviral vectors DBCLS 統合 TV [CC BY 4.0 (http://creativecommons.org/licenses/by/4.0)], via Wikimedia Commons By David Goodsell RCSB PDB Molecule of the Month, CC BY 3.0, https://commons.wikimedia.org/w/index.php?curid=37916906 17

CRISPR technology and Ethics Can edit any cell Use in gene therapy Inexpensive compared to previous methods of creating transgenes or genetically modified animals. Will Cas9 stop cutting? AKA brake failure Gene drives controversial By Mariuswalter Own work, CC BY SA 4.0, https://commons.wikimedia.org/w/index.php?curid=62766590 Other biosafety issues 18

Vaccinations Diphtheria toxin (0.1 µ/kg body weight) used to transport large proteins across cell membranes Pertussis toxin interrupt cell signaling. Seasonal influenza Work with Swine Work with influenza virus (not mouse adapted) Association of Dengue virus pathogenicity and H1N1 Anthrax Field research with elephants, vultures and zebras in Namibia Question 1. G deleted rabies has all of the following qualities except: A. Has a single gene deletion B. Can move from cell to cell transynaptically until it reaches the brain C. Can transfect any synaptically connected neuron if one adds the gene for G protein on a plasmid D. Can be used as a tracer gene by adding jelly fish genes Question 2. PEP for LVV exposure includes A. Acyclovir B. Valcylovir C. Integrase inhibitor D. Protease inhibitor 19

Question 3. The risk of LVV exposure includes all but which answer? A. Insertional mutagenesis B. Insertion of an oncogene C. Recombination to wild type HIV1 D. Dengue Fever 20