Thanks to: || .gov || .edu || .org || || || .com || || || || *==Read===========Write===========Arithmetic 1

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.

3

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

6

Physical Isolation

Marburg, Ebola, Lassa, Crimean-Congo hemorrhagic fever, Smallpox

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.

Non-standard AAs in 5 essential genes: Escape

frequencies.

Zero per trillion

Non-functional UAG attenuates λvir (& T7 ) infection

Lajoie unpublished

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

Esvelt et al eLife

Gene Drives for the Alteration of Wild Populations

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

Gene therapy: 2000 clinical research trials.

18

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

19

Genes Environments Traits (GET) • World’s only open access data sets • Consented for likely re-identification • 100% on Exam – Educate first • Stem Cell Biobank

20 20

CRISPR Human Organoids-on-chips

Ingber, Parker, Knoblich, etc. labs

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.

VUS Variants of Unknown

Significance

27

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