reading & writing omes 5-dec-2019, 12-1 pm, george church ......dec 05, 2019 ·...
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Reading & Writing Omes5-Dec-2019, 12-1 pm, George ChurchDuke Applied Genomics & Precision Medicine Forum
Full COI list: v.ht/PHNc
Lipper 1997Foundation
DOE 1987
2004
2001
@geochurchBlavatnik InstituteRegenesis InstituteSIAT
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Reading & Writing Omes(from the viewpoint of prevention & therapy)
Relevant commercial interests: 3rd Gen Seq: ONT, Roche, Roswell2nd Gen Seq: Nebula Genomics, Veritas Genetics, IlluminaIn situ: Readcoor, BrukerEdit: Editas Medicine, IntelliaTx, CrisprTx, EgenesisTissues in vitro: Cellino, GCTxAging & complex diseases: Rejuvenatebio
(full COI see: v.ht/PHNc)
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3
World’s only fully open access human Genomic, Environmental, Trait data & cells
NIH ENCyclopedia Of DNA Elements11 isogenic cell types.
Critical Assessment of Genome Interpretation
US, Canada, UK, Austria, Korea, China, India
NIST + FDA + PGP = Genomeinabottle.orgStandards: Ashkenazi & Han trios
Jun-2009
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Gene therapies à 2 Low cost alternativesTherapy Source Genes Disease US/yr $Luxturna Spark AAV2/RPE65 LCA-blindness 80 425k/eyeImlygic Amgen HSV1/GM-CSF Melanoma 7200 780k/6moKymriah Novartis CAR(to CD19) Leukemia 9600 475kYescarta Kite Pharma CAR(to CD19) Lymphoma 8000 373kZolgensma Novartis AAV9/SMN1 SMA 500 2.1MSpinraza Biogen ASO SMA 500 4M/10yr Multiplex Rejuvenate AAV/3genes Multiplex 2,800,000 2k ?
1) Common diseases: (age-affected) spread R&D costs2) Rare diseases: Prevention via dating/matchmaking
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1E-3
1E-2
1E-1
1E+0
1E+1
1E+2
1E+3
1E+4
1E+5
1E+6
1E+7
1980 1985 1990 1995 2000 2005 2010 2015
Oligo bp/$
Seq bp/$
Nucleic Acid Read / Write improving exponentially 2-10-fold better than Moore’s law
10 million-fold+
Multiplex NGS in 2003 Multiplex Chip-Syn 2004
$0
1984 HGP haploid1E-2 error rate
2009-12 diploid PGP1E-7 error rate
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Molecular & cell multiplexing1E2 to 1E12 reactions per droplet(not automation or parallelism)
1874 US # 209,241 Quadruplex telegraph. Edison.1987 US #4,942,124 Multiplex sequencing. 2003 Mitra et al. Fluorescent in situ Sequencing on Polymerase Colonies. Analyt. Biochem. (FISSEQ)2004 Tian et al. Accurate Multiplex Gene Synthesis from Programmable DNA Chips. Nature.2009 Multiplex Automated Genome Engineering. Nature2018 Chan et al. Multiplexed testing of pooled donor cells using SNPs and NGS. Genome Medicine
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Addition, subtraction, precise-editing, epigenetics
- Nucleases (error-prone)
e De-aminases CàT, AàG
+-e Recombinases & Primer-Editor (precise & flexible)
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Recombinases & Integrases
l Red recombinase: Gabe Filsinger & Tim Wannierfound interaction with Single-strand binding proteins (SSB)No custom protein or RNA (just donor DNA)
Small S/Y integrases: Ben Weinberg. ML-Syn Custom protein (no RNA)
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GP-Write: precise hyperediting (MAGE)4 Million bp Genome Recoding: 63 & 57 codon types
321 (2013-5) à 62,214 codons (2016-9)
Science 2016 Ostrov, et alNature 2015 Mandell et al
Science 2013 Lajoie et al2. Genetic &Metabolic Isolation
3. Multi-Virus resistance
1. Non-standard amino acids (NSAA)
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2 +1 codons(Fredens..Chin
2019)
6 +1 codons
Genomically Recoded
Organisms (GRO)
4 Mbp synthesized 3kb à 50 kb
Ostrov, Norville, Guell, .. Church 2016
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Ref. Disease Clinical OrganoidOnset
2014 Cardiomyopathy 1y 4 wk2018 Late onset Alzheimer’s 70y 2 wk2019 Schizophrenia 20y 4 wk2019 Bipolar 20y 4 wk
Wang, … Pu (2014) Nature MedicineMeyer … Yankner (2018) Cell Reports.
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REST and Neural Gene Network Dysregulation in iPSC Models of Alzheimer’s Disease
Meyer K, Feldman HM, Lu T, Drake D, Lim ET, King-Hwa L, Bishop NA, Pan Y, Seo J, SuSC, Church GM, Tsai LH, Yankner BA (2018) REST and Neural Gene Network Dysregulation in iPSC Models of Alzheimer’s Disease. Cell Reports.
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The most common disease (affects rare & complex diseases) à 9 Pathways
Restoring mitofunction
Caloric restriction
Reactivate proteinregulation
Epigenetic drugs
Reducing cells withgenomic damage
Telomerereactivation
Stem cell addition
Inflammation reduction, blood rejuvenation
The Hallmarks of Aging Carlos López-Otín, et al. Cell 2013
Clearance of senescent cells
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Non-cell autonomous aging reversal gene therapies
GenAgeThe Ageing Gene Database305 Human, 126 Mouse838 Worm,170 Fly
João Pedro de Magalhães
Noah Davidsohnet al. PNAS Nov-2019
1. High fat diet obesity2. Type 2 Diabetes3. Osteoarthritis4. Cardiac damage recovery5. Kidney disease
à 5 aging diseasesvia combinatorial AAV gene therapy
à
FGF21: glucose handling, sTGFβR2: Soluble formαKlotho: intracellular calcium, serum mineral-ion homeostasis
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Protein Design (AAV & Ab): Multiplex deep sequence testing
DNA Synthesis
DNA sequencingSelection
YY Y
Y
Y
Y
Capsidpackaging
Immune evasion
In vivo delivery
Tissuespecificity
Machine Learning
Eric Kelsic
Pierce Ogden Science Nov 2019
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Mapping the space of tissue tropism modifications
Kidney
Blood Heart Liver
LungSpleen
Position570 605
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VUS-pipeline: Full Transcription Factor Library Collaborators
1,576 genes à 1,748 human TF ORFs. Gateway-compatible vectors (+1200 Sheynkman)
1. Many cell types: Screen for loss of stem cell identity, TRA 1-60
2. Make specific cell types via transcriptome targeting
Ng, Khoshakhlagh, Shipman, Swiersy, Appleton, Huang, Saylor, Trono, Taipale, Hill, Vidal, Busskamp, Church (2017) in prep
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Oligodendrocyte Myelin wrapping Axons
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Human organoids in mice
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Pig organs in primates. Multiplex (43-plex) germline editing
Sugars: GGTA1, CMAH, β4GalNT2 Clotting: human TFPI, TBM, EPCR, vWFMHC: HLA/SLA class I, IIImmune functions: human CTLA4-Ig, HLA-E/G/Cw3 inhibit NK cellsComplement: regulatory genes CD46, CD55, CD59Porcine ERVs: 25 in normal fibroblasts
Luhan Yang, et al 2015 ScienceDong Niu, et al. 2017 Science
MGH: Jim Markmann
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Genome engineering for enhanced organsResistance to: pathogens, senescence, cancer, immunity, cryopreservation, DNA-damage
Lithobates sylvaticus N.Amer. wood frog -14oCSpermophilus parryii, Arctic squirrel -3oCGynaephora groenlandica, Arctic moth -60oCPolypedilum vanderplanki, arid midge 3oKTardigrada, broadly distributed 3oK
Glucose + ureaTrehalose, Late Embryo Abundant, anti-oxidant, heat-shock proteins, AFPs, PUFAs, polyamines, etc.
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DNA-nick toxicity: bFGF & PTFαpreserves highly modified cells
Absence Presence
Niu et al. 2017 Science
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Multiplex Editing Genome-wide
Repeats involved in:Wang et al. Cell Cycle 2011 cancer (Cen, Alu)DeCecco et al. Nature 2019 senescence, inflammation (L1)Singer et al. Trends Neurosci. 2010 neurogenesis (L1)
Category #/ hum cell Length Total bp Previous mutation attemptsERV 25 7500 2.4E+7 Cas9 Yang .. Science 2015, 2017Telomeres 46 8000 3.7E+5 TERT Ramunas .. FASEB J 2015rDNA repeats 300 43000 1.3E+7 I-CreI Paredes .. Genetics 2009
UCE 855 200 1.7E+5 Cas9 Schneider, Biorxiv 2019LINEs 26,000 7000 1.8E+8 dABE Smith .. Biorxiv 2019 Centromeres 1.0E+6 171 1.7E+8 Cas9 Adikusuma .. Mol Ther 2017SINEs (Alu) 1.5E+6 280 4.2E+8 Natural variantsSSR 3.0E+6 16 4.8E+7 Cas9 [14] Monteys..Mol Ther 2017Triplex sites 1.7E+7 20 3.4E+8
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Enabling large-scale genome editing by reducing DNA nicking & off-target
5’-ATTCTACCAGAGGTACAAGGAGG-3’5’-ATTTTATTAGAGGTACAAGGAGG-3’5’-ATTCTGCCAGAGGTACAAGGAGG-3’
Deamination CàTAàG
Cory Smith
1. No nick, dCas92. No MMR3. No U-glycosylase4. Anti-apoptotic molecules 5. Growth factors (bFGF)
Oscar Castanon
Rees H, Liu DR (2018) Base editing: precision chemistry on the genome and transcriptome of living cells. Nat Rev Genet.
Smith CJ, Castanon O, Said K, Volf V, Khoshakhlagh P, Hornick A, Ferreira R, Wu CT, Güell M, Myllykallio H, Church GM (2019) Biorxiv
Verena Volf
Khaled Said
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250
10
20
30
40
50
% o
f rea
ds w
ith ta
rget
ed
Nuc
leot
ide
conv
ersi
on
CBEControlDay 14
nCBE4-gamHL1gR4Day 14
dCBE4-gamHL1gR4Day 14
nABEHL1gR4Day 14
dABEHL1gR4Day 14
ABEControlDay 14
Nick-less deaminase targeting of LINE-1 in human 293T cells (PGP1 iPSC
dABE: 50.61 % x 26100 LINE-1 = 13,470 edits(27,000 diploid)
dCBE: 22 % x 26100 LINE-1 = 5742 edits
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Storing data in DNA <455 ExaBytes/g
Church, Gao, Kosuri 2012 Science
Activity, Behavior, Development
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12 billion cells * 40 mutant gRNAs à 4 trillion bits
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Recording in DNA in vivo: Developmental lineage4 trillion bits stored
Kalhor, Mali, Church 2016 Nature Methods2018 Science
gRNASin situ
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Multiplex cell analysis instruments
nm Plex Instrument
90,000 >1000 NA Spatial Transcriptomics
5,000 40 Ab CyTOF (flow)
250 50 Ab CyCIF (in situ)
250 >1000 NA+Ab ReadCoor (in situ) targeted or un
12 >1000 NA+Ab Bruker Vutara (in situ) targeted or un
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Normal, synthetic, pathological tissues
in situDNA, RNA, protein
Fluor microscopy(hyb, lig, pol)
Multi-axis stages & thermocyclers
FluidicsFluidics
Objective
Spinning Disk Confocal
Novaseq$985k
Vutara$400k
ReadCoor (Church lab)&
Bruker Vutara (Wu lab)12 nm precision biplane & lightsheet
ß 1000-fold thicker
samples than à
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Subcellular& multicellularin situ RNA sequencing PGP1 fibroblasts
312
Lee, Daugharthy, .. ChurchScience 2014, Nature Protocols. 2015
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Walking along chromosomes with super-resolution imaging, contact maps, and integrative modeling. PLoS Gen 2018
Huy NguyenShyamtanuChattorajGuy Nir Brian
Beliveau
Ting Wu, Peng Yin, Erez Lieberman-Aiden, Marc Marti-Renom
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19
19
Chr. 19 step sizes: 1300 to 400 kbChr. 5: all steps are 250 kbChr. 3: all steps are 500 kb
, Perez-Wu 2018
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19
19
33
5
5
Wu 2018
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chr19PGP1f
1 µm1 2 3 4 5 6 7 8 9Nir, Farabella, Perez-Estrada, Ebeling … Wu 2018
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10 270 10 kb 100 nm
OligoSTORM
Nir, Farabella, Perez Estrada, Ebeling … Wu 2018
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10 270 10 kb
100 nm
OligoSTORM
13.40 13.69Mb
Nir, Farabella, Perez-Estrada, Ebeling … Wu 2018
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38
Compile barcode bits Chr.235
1619X
90 Mb
600 kb-1Mb/target (876 kb average)5,000 Oligopaints oligos/target7-70 Mb between targets
Huy NguyenShyamtanu ChattorajDavid Castillo/ Marc Marti-RenomTing Wu
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Oligonucleotide-labeled Protein Recognition
.
hybribody.com/contenu/synthetic-vhh-library-menu/take-advantage-of-vhh-antibody-properties
4x2 nm
ACGTACGTA
DNA Exchange Imaging (DEI): Super-resolution& Multiplex ML-Syn of nanobodies
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Proteins: in situ multiplexing with DNA-Exchange
Wang, Woehrstein, Donoghue, Dai, Avendaño, Schackmann, Zoeller, Wang, Tillberg, Park, Lapan, Boyden, Brugge, Kaeser, Church, Agasti, Jungmann, Yin (2017) Nano Lett.
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Binder library & assay design
Binder LibraryTarget Library Barcode2 differentTarget Libraries• Cytokines• Transcription Factors 6 different
Binder Libraries• Adnectins• Affibodies• Affimers• Alphabodies• Fynomers• Nanobodies
NGS barcodes to see what interacts
Target – Binder Interaction
Reporter
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Machine learning on many Binder-Target interactions
selection
Gleb Kuznetsov & Pierce Ogden
• Adnectins• Affibodies• Affimers
• Alphabodies• Fynomers• Nanobodies
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PGP collaborators NIH NHGRI CEGS
Center for Genomically Engineered Organs
Boyden, Church, Lewis, Wu
NIH Transformative AwardsMolecular RecordingBoyden, Kording
Human iPSC: Alzheimer’sSchizophrenia & BipolarYankner
arep.med.harvard.edu/gmc/ELSI_gc.html pged.org
DARPA, NSF, DOE GP-WriteIsaacs, Wang, Silver, Way, Boeke
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Mammalian Genome Recoding Team
Yuting Chen
Kevin YangStephen Casper
Harvard Undergrads
Richie Kohman
QFASTR
SIAT
Eriona Hysoli
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Germline vs Somatic Gene Therapy
1. Billion-fold lower off-target (due to cell #)
2. Clonality allows checking (additional million-fold)
3. Immune Tolerance
4. 100% Delivery to every cell
5. $0 to subsequent generations
Disadvantages:
1. Very long term clinical trials >20 years
2. 130 newborns/yr vs 7.7 billion total
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Non-functional UAG thwarts 4 / 6 Viruses
Ma & Isaacs (2016) Cell Syst. 3: 199–207.
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Carbon sequestration (not just slowing losses)
Human use: 10 Gton/yr Tundra soil carbon: 1,400 GtonAtmosphere: 850 Gton Tropical forests: 375 Gton
4-Jul-201720132012
Nicholas Wade
2008
1. Alternatives to meat2. Virus-resistant algae, segregate CHO from Fe/P3. Keystone species to switch back trees to grass
increase albedo, photosynthesis, winter snow pack
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Yakutia branch of Russian Academy of Sciences Photos: GC
medium.com/@eriona.hysolli/an-american-russian-collaboration-to-repopulate-siberia-with-woolly-mammoths-or-something-similar-9cbac4e985cb
USDA per 100 grams Bison = 109 calories, 1.8 grams fat Beef = 291 calories, 24 grams fat
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Targeted Cytosine Deaminases
2009-2011: Fusion Peptides That Bind to and Modify Target Nucleic Acid Sequences. Church & Yang US Pat. App. 20110104787.
2016 Programmable editing of a target base in genomicDNA without double-stranded DNA cleavage. Komor … Liu Nature CàT Cas9-BE
2016 Engineering and optimising deaminase fusions for genome editing. Yang … Church. Nature Comm.
2019 Cytosine base editor generates substantial off-target SNVs in mouse embryos. (20-fold APOBEC1 vs TadA) Zui … Yang. Science
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Adenosine Deaminase2017 Programmable base editing of A•T to G•Cin genomic DNA without DNA cleavage. Gaudelli … Liu. Nature
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Reverse Transcriptase
2019 Search-and-replace genome editing without double-strand breaks or donor DNA. Anzalone … Liu. Nature