veränderbarkeit des säugetiergenoms durch crispr-cas: … · 2017-10-11 · veränderbarkeit des...
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Veränderbarkeit des Säugetiergenoms durch Crispr-Cas:
Was wissen wir und was können wir erwarten
Franz Hofmann, Pharmakologie, TU München
Biologentag 2017 des VBIO-NRW: Genom-Editierung und GentherapieEdierung
Klassische Gen-Modifizierung mit dem Cre-lox-System
Embryonale Stammzellen
Identifizierung der Zelle, die das mutierte Gen enthält
Transfektion mit Cre und Entfernung der NeoTK Kassette
Identifizierung der Zelle, die das mutierte Gen enthält
Vermehrung dieser Zelle und Injektion der Zellen in Blastozyste
Selektion des Babies mit dem gewünschten GenotypBrandmayr et al J Biol Chem. 287(27): 22584 (2017)
Die neuen programmierbaren Systeme
ZFN Zinkfinger-Nukleasen
TALEN Transcription activator-like effector nucleasen
CRISPR/Cas9-System clustered regularly interspacedshort palindrome repeats /CRISPR-associated endonuclease
Jackson et al., Science 2017 356, eaal5056 7 April 2017
CRISPR-Cas adaptation and defense.
L = leader sequence
R = repeat squences
Color = spacer = foreign sequence
Doudna & Charpentier SCIENCE 346: 1077 (2014)
Protospacer adjacent motif (PAM)
5'-NGG-3'
Nukleasen mit verschiedenen PAM-
Spezifitäten
Type I = mehrere kleine Cas-Proteine
Cas 3 dsDNA
Type II = Cas 9 dsDNA
Type IIIA = Csm6 dsDNA
Type IIIB = Cmr4 RNA
Komor et al (2017) Cell 168: 20
Komor et al (2017) Cell 168: 20
Modified from SCIENCE 346: 1077 (2014)
Examples of cell types and organisms that have been engineered using CRISPR/Cas9
Modifizierung Humaner Gene in vitroBlutzellen und Blutstammstellen
Transfektion der Zellen mit Crispr/Cas
Vermehrung der transfizierten Zellen
Reinfundierung der modifizierten Zellen
Gene Editing of CCR5 in Autologous CD4 T Cells of Persons Infected with HIV
Pablo Tebas, M.D., David Stein, M.D., Winson W. Tang, M.D., Ian Frank, M.D., Shelley Q. Wang, M.D., Gary Lee, Ph.D., S. Kaye Spratt, Ph.D., Richard T. Surosky, Ph.D., Martin A. Giedlin, Ph.D., Geoff Nichol, M.D., Michael C. Holmes, Ph.D., Philip D. Gregory, Ph.D., Dale G. Ando, M.D., Michael Kalos, Ph.D., Ronald G. Collman, M.D., Gwendolyn Binder-Scholl, Ph.D., Gabriela Plesa, M.D., Ph.D., Wei-Ting Hwang, Ph.D., Bruce L. Levine, Ph.D., and Carl H. June, M.D
N Engl J Med 370:901-910, 2014
N Engl J Med 2017;376:848-55.
Gene Therapy in a Patient with Sickle Cell Disease
Jean-Antoine Ribeil et al
Sickle cell disease results from a homozygous missense mutation in the β-globin
gene that causes polymerization of hemoglobin S. Gene therapy for patients with
this disorder is complicated by the complex cellular abnormalities and challenges
in achieving effective, persistent inhibition of polymerization of hemoglobin S. We
describe our first patient treated with lentiviral vector–mediated addition of an
antisickling β-globin gene into autologous hematopoietic stem cells. Adverse
events were consistent with busulfan conditioning. Fifteen months after treatment, the
level of therapeutic antisickling β-globin remained high (approximately 50% of
β-like–globin chains) without recurrence of sickle crises and with correction of the
biologic hallmarks of the disease.
N Engl J Med 2017;376:848-55.
Non-Clinical development of in vitro and in vivo gene editing
Target
CCR5; HIVHBB; β-ThalassemiaDMD; Duchenne muscular dystrophyG6Pase; Glycogen storage disease type IACFTR; Cystic fibrosisPD-1; Cancer; melanoma
Als Nuklease wird benutzt ZNF, Talen, Crispr-Cas9
Strategies for In Vivo Delivery of
CRISPR-Based Genome-Editing Agents
Komor et al (2017) Cell 168: 20
Immuntherapie von Krebs
Prinzip: CAR-T-Zellen = Chimeric Antigen Receptor T-Zellen for advanced Therapies
Erfolgreich bei einigen hämatologischen Tumoren
Problem: Umgehung des Immunsystems / des Körpereigenen Abwehrsystems
Probleme: ZytokinsturmB-Zell Runterregulierung / AntikörpermangelKosten 50 -70 000 € pro Patient
Antibody-modified T cells: CARs take the front seat for hematologic malignanciesMarcela V. Maus, Stephan A. Grupp, David L. Porter and Carl H. JuneBlood 2014 123:2625-2635;
Tisagenlecleucel-T (Novartis) Therapie von B-Zell-akuter lymphoblastischer Leukämie (ALL)
Axicabtagene ciloleucel (Kite Pharma Inc.) Therapie von refractory diffuse large B cell lymphoma (DLBCL), transformed follicular lymphoma and primary mediastinal B cell lymphoma
An immunogenic personal neoantigen vaccine for patients with melanoma
Patrick A. Ott…& Catherine J. Wu: Nature 547,217;2017
Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer
Ugur Sahin … & Özlem Türeci: Nature 547, 222;2017
Melief CJM Nature 547, 165; 2017
Programmed cell death protein 1, PD-1 and/or CD279
CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells
from cancer patientsShu Su et al Scientific Reports 6, Article number: 20070 (2016)
First trial of CRISPR in people Chinese team approved to test gene-edited cells in people with lung cancer.
Nature 4 7 6 | N AT U R E | VO L 5 3 5 | 2 8 J U LY 2 0 1 6
First CRISPR clinical trial gets green light from US panel
The technique's first test in people could begin as early as the end of the year.
Nature 2016
Programmed cell death protein 1, PD-1and/or CD279
Clinical trials of gene editing
PDCD-1 , Gene for Programmedcell death protein 1, PD-1and/or CD279
SHIM et al APS (2017) 38: 738–753;
Problems associated with Gene Therapy
Off-target genotoxicity
Immunogenicity of Nucleases and Vectors
Pharmacokinetics and biodistribution
Tumorgenicity
Safety of delivery system
Efficacy
Duration of gene editing
Animal models for efficacy tests
Purity and Sterility of gene-editing therapeutics
Manufactoring processes
Sperm injection Cas9 mRNA/sgRNAs injection
Culture9 hr
Embryo transfer
Mutant founders Surrogate mother
Gestation5 months
http://dx.doi.org/10.1016/j.cell.2014.01.027Niu et al. (2014) “Generation of Gene-Modified Cynomolgus Monkey via Cas9/RNA-Mediated Gene Targeting in One-Cell Embryos”
CRISPR-Cas9 modifies your DNA
Niu et al. Cell 156, 836–843, 2014
Modify the Mammalian Genom
The Belgian Blue has a natural mutation in the myostatin gene which codes for the protein, myostatin ("myo" meaning muscle and "statin" meaning stop). Myostatin is a protein that acts to inhibit muscle development.
A Belgian Blue Bull
Phenotype sharedby cattle known as Piedmontese
(https://en.wikipedia.org/wiki/Belgian_Blue#cite_note-Kambadur-4)
Not yet 5, he can hold seven-pound weights with arms extended, something many adults cannot do.
Myostatin-KO Pigs created in South Korea
„Super-muscly pigs created by small genetic tweak” NATURE 523, 13-14, 2015
Ziel Gen-Defekte bei einem Menschen korrigieren!
In Deutschland verboten!
Geht nur bei einem Embryo im 1-Zellstadium (Zygote).
Im anglo-amerikanischen Forschungsbereich ist das Experimentieren an menschlichen Embryonen bis zum 14 Tag nach Fusion von Spermium und Oocyte (Zeugung) erlaubt.
Korrektur eines defekten Genes
PID erlaubt die Sortierung von 3 oder 5 Tage alten Embryonen nach ~200 spezifischen Gendefekten, wenn es von einer Ethikkommission genehmigt wird.
CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and miceYu Zhang, Chengzu Long, Hui Li, John R. McAnally, Kedryn K. Baskin, John M. Shelton, Rhonda Bassel-Duby, Eric N. Olson
Sci. Adv. 2017;3: e1602814
Repair of Duchenne Muscular Dystrophy (DMD)
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McNally & Wyatt (2017) Circulation. 136:979-981
Eteplirsen (EXONDYS 51 ®)
CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein
Tang, L., Zeng, Y., Du, H. et al. Mol Genet Genomics (2017). doi:10.1007/s00438-017-1299-z
Previous works using human tripronuclear zygotes suggested that the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system could be a tool in correcting disease-causing mutations. However, whether this system was applicable in normal human (dual pronuclear, 2PN) zygotes was unclear. Here we demonstrate that CRISPR/Cas9 is also effective as a gene-editing tool in human 2PN zygotes. By injection of Cas9 protein complexed with the appropriate sgRNAs and homology donors into one-cell human embryos, we demonstrated efficient homologous recombination-mediated correction of point mutations in HBB and G6PD. However, our results also reveal limitations of this correction procedure and highlight the need for further research.
Human 3PN Embryos; nicht lebensfähig
Indel efficiency HDR efficiency NHEJ efficiencyRAG1 60% 60%NEK1 70% 69%G6PD 70% 5% 10-20% HBB 5% 10%
Human 2PN Embryos; Gesund normal
ß-Thalassemia; stop codon at the 59th codon of HBB
male sperm heterozygous for ß-thalassemia + normal human oocytes
4 embryos that are heterozygous
2 embryos with indels from them 1 embryo with correction of ß-thalassemia HHB gene.
G6PD Mutation G1376T correction apparently more efficient (100%)
Indel = Insertionen und Deletionen
Human β-globinGlucose-6-Phosphate-DehydrogenaseNIMA (never in mitosis gene a)-related kinase 1
Recombination activating gene 1
WINBLAD N & LANNER F Nature 548; 398 (2017)
Correction of a pathogenic gene mutation in human embryos
Ma H, …….. & Mitalipov S Nature in press (2017) MYBPC3 gene: 42 of 58 embryos tested (72.4%) were corrected
Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro
Sarah Ellys Harrison, Berna Sozen, Neophytos Christodoulou, Christos Kyprianou, Magdalena Zernicka-Goetz
Science 14 Apr 2017: Vol. 356, Issue 6334, pp. 137-138
In vitro stem cell–embryos model mouse embryo development from implantation to gastrulation.
naturel
Embryonic stem cells +Extraembryonic trophoblast stem cells
JOHN AACH, JEANTINE LUNSHOF, ESWAR IYER AND GEORGE M CHURCH
Addressing the ethical issues raised by synthetic human entities with embryo-like features
eLife 2017;6:e20674.
The "14-day rule" for embryo research stipulates that experiments with intact human
embryos must not allow them to develop beyond 14 days or the appearance of the primitive streak.
However, recent experiments showing that suitably cultured human pluripotent stem cells can selforganize
and recapitulate embryonic features have highlighted difficulties with the 14-day rule and
led to calls for its reassessment. Here we argue that these and related experiments raise more
foundational issues that cannot be fixed by adjusting the 14-day rule, because the framework
underlying the rule cannot adequately describe the ways by which synthetic human entities with
embryo-like features (SHEEFs) might develop morally concerning features through altered forms of
development. We propose that limits on research with SHEEFs be based as directly as possible on
the generation of such features, and recommend that the research and bioethics communities lead a
wide-ranging inquiry aimed at mapping out solutions to the ethical problems raised by them.
Figure 1. Biological landscape of embryos and Synthetic human
entities with embryonic features (SHEEFs) in relation to moral
status. Embryos derived through sexual intercourse or assisted
insemination (Cantineau et al., 2013; Hurd et al., 1993) (left),
cultured embryos (center), and SHEEFs (right) start from types
of pluripotent cells (zygotes and hPSC; bottom) that have
different capacities for development: Embryos formed from
zygotes derived sexually can develop into fetuses in utero
(vertical arrows, left). Embryos can also be generated from
zygotes formed in vitro
Aach et al. eLife 2017;6:e20674.
vector-borne diseases MosquitoesErkrankung
Malaria
Dengue
Zika
Unterbindung der Übertragung des Vektors z.B. Expression eines Gens, das den Vektor zerstört
Wildtyp Mosquito in einen Vektor-zerstörenden Mosquito umwandeln
Gene Drives
Valentino M. Gantz and Ethan Bier. The mutagenic chain reaction: A method for converting heterozygous to
homozygous mutations. SCIENCE 348:442 – 444, 2015
Oye et al. Regulating gene drives. SCIENCE 345: 626 – 628, 2015
Gene Drive
Prinzip eines Gene Drive Konstrukts
Champer et al NATURE REVIEWS | GENETICS 17:146-159; 2016 |
A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria
mosquito vector Anopheles gambiaeHammond et al. NATURE BIOTECHNOLOGY 34: 78-83; 2016
Inactivation of 3 genes recessive female-sterility phenotype
US National Academies Hit the Brakes on Gene Drive–Modified Organisms 2016
Highly efficient Cas9-mediated gene drive for population modification of the malaria vector
mosquito Anopheles stephensiGantz et al. PNAS E6736–E6743;2015
Expression of two antibodies that target the human malaria parasite Plasmodium falciparum ookinete protein Chitinase
1 and the circumsporozoite protein (CSP). Prevention of sporocoites and, therefore, were incapable of transmitting
parasites
Möglicher Gebrauch von Gene-Drives
Esvelt et al. eLife 2014;3:e03401.
Esvelt wird gefördert für Development of Gene-DrivesVon US Defense Advanced Research Projects Agency(DARPA)
Novel CRISPR/Cas9 gene drive constructs revealinsights into mechanisms of resistance allele formation and drive efficiency in geneticallydiverse populations.Champer J, …….Messer PW.
Plos Genet.:2017 Jul 20;13(7):e1006796.
In a heterozygous female with genotype D/+, expression
of Cas9 in a germline cell can produce one of three outcomes: (i)
successful conversion of the wild type allele into a drive
allele by HDR, (ii) formation of a resistance allele when HDR is
incomplete or cleavage is repaired by NHEJ, or (iii)
continuing presence of the wild type allele if no cleavage occurred or
was perfectly repaired. For our nanos construct in
the w1118 line, we observed successful germline conversion in D/+
females at a rate of approximately 60%, leaving 80%
of gametes with gene drive alleles. Almost all remaining gametes
(20%) contained resistance alleles, with only less than
1% of gametes carrying a wild type allele.
Question:
Who transfers the first gene-corrected human zygote to a pseudo-pregnant female?
Which country allows that?
A bacteriophage encodes its own CRISPR/Cas adaptive response to evade host innate immunityKimberley D. Seed, David W. Lazinski, Stephen B. Calderwood, and Andrew Camilli,*
Nature. 2013 February 28; 494(7438): 489–491.
Use natural or engineered bacteriophages that encode Crispr/Cas to destroy bacterial resistance genes for antibiotics
Problems: Phages are strain or even substrain specific Bacteria could evolve resistance to phages Engineered phages have not been tested in humans Phages may trigger immune response in humans Phages could transfer antibiotic-resistance genes to non-resistant bacteria
Scheufele, DA et al. „U.S. attitudes on human genome editing.“ Science 357, 553 (2017)
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