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Biocontainment of GMOs 13-Oct-2015 10:50-11:50 AM ABSA Providence, RI
LSRF
NHGRINIGMS
Azco
Oppenheimer Foundation
ABSA American Biological Safety Association
Physical, Species, Genetic, Metabolic, Ecological Isolation
• Exponential changes in biology • Biocontainment for bacteria • Zoonoses & endogenous viruses • Gene-drives for vectors & invasives • Gene therapy safety issues.
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5-10X/year since 2005 (vs 1.5X Moore’s Law)
Carr & Church, Nature Biotech
Synthetic biology
Sequencing
Isolation
Testing
Redundant systems
Licensing
Safety Enginering Standards & modeling
Surveillance
& the costs of doing nothing
Safety via Surveillance of Synthetic DNA
Training & scenario brainstorming Communication & transaction surveillance • Dropping costs of DNA monitoring • Phosphoramidites & instruments
Church GM (2004) A Synthetic Biohazard Non-proliferation Proposal Church GM (2005) Let us go forth and safely multiply. Nature 438: 423 Bügl, et al. (2007) DNA synthesis and biological security. Nature Biotech Church GM (2009) Safeguarding Biology. Seed 20:84-86. Church GM (2010) Presidential Commission for the Study of Bioethical Issues
Physical, Species, Genetic, Metabolic, Ecological Isolation Order of construction matters.
Science 2013: Lajoie, et al. Nature 2015 Mandell et al. 2. Genetic isolation
3. Metabolic Isolation
4. Multi-Virus resistance
1. Non-standard amino acids (NSAA)
13 codons
7 codons 3 New AA
Genomically Recoded
Organisms (GRO)
4 Mbp synthesized 3kb 50 kb
Ostrov, Norville, Guell, et al.
Humanized for xeno transplants &
Lower zoonoses GGTA1, CMAH, SLA/HLA Complement, Clotting 62 PERVs = Porcine Endogenous RetroViruses Exogenous PCMV, PEDV, PRRSV
1.7 million sufferers of end stage kidney disease (ESKD) worldwide Bendorf et al. 2013
Esvelt, Smidler, Catteruccia, Church July 2014 eLife
Safer Gene Drives for the Alteration of Wild Populations
Physical, Species, Genetic, Ecological Isolation
Physical, Species, Genetic, Metabolic, Ecological Isolation
Esvelt KM, Smidler AL, Catteruccia F, Church GM (2014) eLife
Intentional (de) extinction: Risks & Benefits
• Cost, other species • Gene drives restricted to wild sexual species
Intentional extinction examples Past: Smallpox Future: Malaria, Polio
Intentional de-extinction examples Past: 1918 flu, HERV Future: Mammoth
AIDS clinical trial Inactivating both copies of the HIV1 coreceptor gene
CCR5 T-cell
HIV Risk reduction using precise genome editing
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Safety for shareable human genomes/cell standards
NIST + FDA Genomeinabottle.org PersonalGenomes.org Cohort consented for re-identification & commercial use. 8 Trios.
5 Sites: Boston, NYC, Toronto, London, Vienna 100,000 genomes each.
CAGI, NIH ENCODE, NCBI, Coriell Human SynBio resources : BACs, CRISPR, Cells
GET-evidence.org pgEd.org
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Genes Environments Traits (GET) • World’s only open access data sets • Consented for likely re-identification • 100% on Exam – Educate first • Stem Cell Biobank
Gene Therapy Risks
Blood 1996 Hacein-Bey et al. Science 2003 Hacein-Bey-Abina et al. 9 of 10 patients cured of SCID-X1 with 4x108 cells 2 of 10, after 3 years, developed exponential clonal proliferation of mature T cells (LMO2 oncogene)
MFG: Moloney murine leukemia virus LTR vector
Not all off-targets are equal. One cell can be better than many.
Tsai, Joung et al. GuideSeq, best gRNA: RFN2 30 to 105 genomes * 3 x 109 bp = 1011 to 3 x 1014 bp Does not include specificity improvements 3 kbp mutation window * 1207 Tumor Suppressors (1 hit) Single sperm: 10-4 to 10-8 haploinsufficient mutations. 0.1% body: 1010 cells: 106 to 100 TS mutations / patient
Haploinsufficient = ~3000 journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1003484 bioinfo.mc.vanderbilt.edu/TSGene #cells: pubmed/23829164
Improving specificity
Theoretical & empirical gRNAs & dose optimization Mali et al, Cong et al (Church, Zhang labs) Science 3-Jan-2013 Dimeric nucleases. Mali, et al, Ran et al (Church, Zhang labs). Nat Biotechnol. 1-Aug-2013. Cell 12-Sep-2013 Tsai et al, Guilinger et al (Joung, Liu) Nat Biotechnol. 25-Apr-2014 Truncated guide RNAs. Fu, et al. (Joung lab). Nat Biotechnol. 26-Jan-2014
Why Somatic Gene Therapies?
Current Practice • Preconception tests • IVF-PGD • Prenatal tests (NIPT) • Newborn tests • Preventative environment, nutrition, surgery • Conventional therapies (chemical, protein, RNA)
Subset of de novo mutations, missed by the above & when post-natal therapy is not too late developmentally.
Why Editing? Subset of above involving dominant deleterious alleles (not treatable by inhibitory therapies) Integrases have better HR : NHEJ than CRISPR.
Why Human Germline Modifications?
Case 1) Both parents have only disease-causing DNA variants. This can occur in the one fifth of marriages worldwide which are consanguineous. This situation is not addressed by PGD or NIPT. Treating sperm such that they no longer carry the disease variant could be an option. Post-natal therapy is too late developmentally. Case 2) Post-natal gene therapy risk of off-target effects, especially cancer. Risk goes up with the number of cells treated. Thus, treatment of one sperm cell could be a billion times less risky than the treatment of a billion somatic cells after birth.
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N=1, Correlation Causality Animal (humanized) testing
Enhanced muscle growth, decreased body fat & decreased atherosclerosis (2009 Endocrine Society, Bhasin, et al BU)
Myostatin double nulls Germany
mouse
MSTN -/-
Barth syndrome
• Cardiomyopathy
• Defective cardiolipin composition
• TAZ mutation
Wang, McCain, Yang, Pu, Parker, et al. Nature Medicine 2014
PGP1-NT AACCATG-------------GGGACTGGGTG 0 bp PGP1-BTHH AACCATG--------------GGACTGGGTG -1 bp PGP1-NHEJ AACCATGagaagctaaccatgGGACTGGGTG +8 bp
Patient mutations normal hiPSC cardiomyocytes
TAZ mutation is responsible for cardiomyocyte morphology abnormality (edited PGP iPSC)
Reduced sarcomere organization -- complemented by mRNA
Wang, McCain, Yang, Pu, Parker, et al. Nature Medicine May-2014
ABSA American Biological Safety Association
Physical, Species, Genetic, Metabolic, Ecological Isolation
• Exponential changes in biology • Biocontainment for bacteria • Zoonoses & endogenous viruses • Gene-drives for vectors & invasives • Gene therapy safety issues.
Space Genetics: Bones, Cancer, Neuro, Microbes
astro.virginia.edu/class/skrutskie/images/weightless.jpg
3ko blastocysts
PERV blastocysts
Unaffected karyotype,
Pig embryos from somatic cell
nuclear transfer
Yang, Güell, Niu, George, Lesha, Grishin, Xu, Poci, Shrock,
Cortazio, Wilkinson, Fishman, Church (Science, in revision)
Synthetic protective alleles. Trade-offs & tuneability
TERT Overprod. Low aging (mice) CDKN2A Overprod. Low cancer (mice) TP53 Overprod. Low cancer (mice) GRIN2B Overprod. High learning & memory (mice, rats) PDE4B Inhib. Low anxiety, high problem solving (mice)
Tomás-Loba A, et al. (2008) TERT, p53, p16, and p19ARF. Wang et al. (2009) Brim BL et al. (2013) GluN2B McGirr A, et al (2015) PDE4B
Further testing in pigs & dogs?
Rare protective alleles. Trade-offs & tuneability LRP5 G171V/+ Extra-strong bones MSTN -/- Lean muscles & low atherosclerosis CCR5 -/- HIV resistance FUT2 -/- Norovirus resistance PCSK9 -/- Low coronary disease SCN9A -/- Insensitivity to pain APP A673T/+ Low Alzheimer’s APOE E2/E2 Low Alzheimer’s (E2=R112C, R158C) GHR,GH -/- Low cancer SLC30A8 -/+ Low T2 Diabetes IFIH1 E627X/+ Low T1 Diabetes ABCC11 -/- Low Odor production
The Resilience Project
Augmentation Somatic gene modifications can spread more rapidly than germline modifications, since only 1% of the population per year (that is births) are of the correct age for germline impact, while somatic therapy is applicable at any age. Furthermore, germline can take decades to have impact, while somatic therapies could have impact in weeks. Pathogen resistance Longevity and cancer resistance Behaviorial & cognitive
Regulation Challenges
The issue may not be scientists, parents, or governments responding to real needs, but financial incentives creating undesirable demand.
Banned EU Convention on Human Rights and Biomedicine: “not to introduce any modification in the genome of any descendants.” NIH “RAC will not at present entertain proposals for germ line alterations” [in human research participants] Parents expose their germline to highly mutagenic environmental sources, such as chemotherapeutic agents, X-ray/CT scans, ethanol, and high altitude. Should intentional exposure to random mutations be more acceptable than engineering changes to be safe and effective?
osp.od.nih.gov/sites/default/files/NIH_Guidelines_0.pdf nih.gov/about/director/04292015_statement_gene_editing_technologies.htm