Genetic modifications: from cells to organisms

Transcription

1 Genetic modifications: from cells to organisms As an example: studying anti-cancer therapy resistance in genetically engineered mouse models (GEMMs) Sven Rottenberg Institute of Animal Pathology Images and graphs are from multiple sources and were partly altered to serve educational purposes. Many images are from Alberts. All material for internal usage during Summer School only. NOT to be distributed outside of Summer School 2017.

2 Cancer is a complex disease Oncogenes Tumor suppressor genes Blood lymph Metastasis Normal cells Primary tumor Immune cells Stroma Angiogenesis WT cells

3 but we have substantial knowledge about what causes cancer

4 and we have powerful weapons to target cancer

5 The painful truth (human medicine) Most cancer patients who have disseminated cancers cannot be cured and cancer is not easily turned into a chronic disease

6 The painful truth (human medicine) Most cancer patients who have disseminated cancers cannot be cured and cancer is not easily turned into a chronic disease The metastatic tumors are eventually resistant to all anti-cancer therapies available

7 The painful truth (human medicine) Most cancer patients who have disseminated cancers cannot be cured and cancer is not easily turned into a chronic disease The metastatic tumors are eventually resistant to all anti-cancer therapies available Therapy resistance remains the major handicap in cancer treatment

8 What about veterinary medicine? Most animals with inoperable, disseminated or locally recurrent cancers are euthanized Systemic anti-cancer therapy is less common

9 What about veterinary medicine? Most animals with inoperable, disseminated or locally recurrent cancers are euthanized Systemic anti-cancer therapy is less common Nevertheless... More and more pet owners want their vets to apply local or systemic anti-cancer therapy to try to cure the animals or at least significantly increase survival

10 The clinical hurdle of anti-cancer therapy resistance will become more relevant in veterinary medicine What about veterinary medicine? Most animals with inoperable, disseminated or locally recurrent cancers are euthanized Systemic anti-cancer therapy is less common Nevertheless... More and more pet owners want their vets to apply local or systemic anti-cancer therapy to try to cure the animals or at least significantly increase survival

11 Some major challenges we are facing To find the right (combination) therapy for the right patient (personalized medicine)

12 Some major challenges we are facing To find the right (combination) therapy for the right patient (personalized medicine) To avoid unnecessary treatments from which the patients will only suffer (predictive markers)

13 Some major challenges we are facing To find the right (combination) therapy for the right patient (personalized medicine) To avoid unnecessary treatments from which the patients will only suffer (predictive markers) To understand and circumvent therapy resistance

14 How do we tackle these challenges?

15 In vitro FUNCTIONAL SCREENS shrnas, CRISPR/Cas9 insertional mutagenesis Drug selection Surviving colonies

16 In vitro FUNCTIONAL SCREENS In vivo CLINICAL SAMPLES shrnas, CRISPR/Cas9 insertional mutagenesis Drug selection Surviving colonies

17 Mouse models as surrogate patients to study treatment responses Genetically engineered mouse models (GEMMs) Patient-derived xenograft (PDX) models Additional mutations Tumors 1 st Intervention Minimal residual disease? Relapses 2 nd Intervention Cross-resistance? Mechanisms? Resistance

18 In vitro FUNCTIONAL SCREENS In vivo CLINICAL SAMPLES shrnas, CRISPR/Cas9 insertional mutagenesis Drug selection Surviving colonies

19 FUNCTIONAL SCREENS CLINICAL SAMPLES shrnas, CRISPR/Cas9 insertional mutagenesis Drug selection Surviving colonies FUNCTIONAL IN VIVO MODELS tumor volume % Genetically engineered mouse models for BRCA1/2- mutated breast cancer days

20 Which mouse model to choose to study drug resistance??

21 Xenografts of human cell lines: the classical way to study tumor responses in vivo Human cancer Tumor cell culture Tumor cell line xenograft Tumor cell line 21 Tumor cell injection IMMUNO- -DEFICIENT ANIMALS Advantage: high reproducibility because of clonal cell; genetic targeting in vitro Disadvantage: loss of morphology, selection of a single clone on plastic which does not represent the original tumor Courtesy of M.F. Poupon

22 There are substantial differences between real tumors in patients and cell line-derived models Real human tumors (ovarian cancer) Cell line-derived models Gillet J et al. PNAS 2011;108:

23 Xenografts of human cell lines: the classical way to study tumor responses in vivo Human cancer Tumor cell culture Tumor cell line xenograft Tumor cell line 23 Tumor cell injection IMMUNO- -DEFICIENT ANIMALS Advantage: high reproducibility because of clonal cell; genetic targeting in vitro Disadvantage: loss of morphology, selection of a single clone on plastic which does not represent the original tumor Courtesy of M.F. Poupon

24 A second choice: Patient-derived tumor Xenograft (PDX) Human cancer tumor xenograft Tumor cell culture Direct tumor transplantation Tumor cell line xenograft Tumor cell line Tumor cell injection IMMUNO- -DEFICIENT ANIMALS Major improvement: morphology is kept, more similar genomic profiles Remaining disadvantages: genetic targeting of tumors is not easy and tumors are in carried in immunodeficient mice Courtesy of M.F. Poupon 24

25 A third choice: reproducing sporadic human cancer in mice by genetical engineering sporadic cancer controlled switching conventional transgenic/knockout controlled switching of multiple mutations Epithelial cell with 1 mutation Mutagen/radiation Epithelial cell with 2 mutations Epithelial cell with 3 or more mutations (invasive) Controlled switching

26 How are transgenic and knockout mice made?

27 The zygote route: Modification of the mouse germ line via transgenesis Oocyte donor DNA injection into fertilized oocytes Implantation into oviduct Tissue-specific promoter Coding sequences with intron Poly-A signal 10-20% transgenic offspring

28 Properties of transgenic mice Transgenesis is quick-and-dirty Quick: transgenic mice can be generated within 1-3 months Dirty: transgene integrates random into the host genome: Expression may depend on integration site Variable number of concatenated transgene copies integrate Integration may disrupt host genes

29 The CRISPR/Cas9 revolution Repair through NHEJ introduces random insertions/deletions, which potentially disrupts the reading frame

30 Background - why is CRISPR/Cas9 so revolutionizing? To really prove a specific mechanism we usually want to target a gene (product) of interest The challenge: How to target a gene? - RNAi: no complete deletion, off-target effects - Gene targeting using homologous recombination (in embryonic stem cells): not very efficient, long procedure, only one allele changed. - Gene trapping (random integration): tedious selection procedure, off-target effects, only one allele targeted - dominant negative mutants: does not always work; same effect as knock-out? - inhibitors / antibodies that target the protein: really specific? CRISPR-Cas9 provides a new tool to target a gene of interest

31 History of CRISPR/Cas9 discovery

32 Repeated elements with common features interspersed by non-repetitive spacers in multiple organisms

33 Coining the term CRISPR, realization of common CRISPR upstream regions and Cas genes Clustered Regularly Interspaced Short Palindromic Repeats

34 Spacers contain phage sequences and strains carrying these are resistant to phages

35 Spacers contain phage sequences and strains carrying these are resistant to phages

36 The spacers can be used to program resistance

37 Bacterial adaptive immune systems Step 1: Programming

38 Bacterial adaptive immune systems Step 2: RNA Transcription and processing

39 Streptococcus pyogenes SpCas9 Type II CRISPR system PAM: Protospacer Adjacent Motif Distinguishes self from non-self In SpCas9: NGG =grna, sgrna

40 Explosion of high-impact papers

41 Cas9/CRISPR knockouts Proper repair by HR or NHEJ Or, when offering repair template (oligo, plasmid), Specific mutations or knock-ins can be made Deleterious repair by NHEJ

42 Additional Applications Multiple grnas Mutant Cas9 / CRISPRi Combined

43 Cell 153, , May 9, 2013

44 The zygote route: Targeted mutations due to CRISPR/Cas9 technology

45 The zygote route: Targeted mutations due to CRISPR/Cas9 technology Creation of frameshift or point mutations in one or more genes simultaneously in an early embryo Introduction of a specific knock-in allele or conditional knockout allele No in vitro steps are required Easy screening of founder animals using PCR or Southern blotting Substantial gain in time: the design and cloning of CRISPR constructs is fast and easy; only in the case of transgene integration does cloning of the accompanying targeting construct demand time

46 The conventional embryonic stem cell (ESC) route: Modification of the mouse germ line via gene targeting in ESCs Blastocyst donor Implantation into oviduct Foster ICM Blastocyst ICM ES cells Injection into blastocysts Chimeric mice ~ ~ Chromosomal DNA x PGK neo pa Targeting vector F1 ~ PGK neo pa ~ Knock-out allele ES cell mice

47 The conventional embryonic stem cell (ESC) route: Modification of the mouse germ line via gene targeting in ESCs Blastocyst donor Implantation into oviduct Foster ICM Blastocyst ICM ES cells Injection into blastocysts Chimeric mice Multiple gene targeting using CRISPR/Cas9 x F1 Cell 153, , May 9, 2013 ES cell mice

48 Properties of targeted mutations via ESCs Targeted mutagenesis via ESCs is slow and precise Slow: generation of mice with targeted mutations takes 6-12 months Precise: mutations are targeted to a defined site in the mouse genome No variation in expression No variation in copy number No variation in integration site

49 Overview of genetic modifications in mice Huijbers et al. Nat Protoc Nov;10(11):

50 Properties of conventional transgenic onco-mice The transgene-promoter determines in which organ tumors will develop: MMTV-Myc: mammary tumors Eμ-Myc: lymphoid tumors Transgenic onco-mice do not mimic sporadic cancer: Transgene is expressed in many cells simultaneously: Transgene is early expressed (often already during embryogenesis) Transgenic mice often develop many tumor simultaneously

51 Limitations of "conventional" tumor suppressor (TS) gene knockouts Many TS gene knockouts are embryonic lethal (e.g. Rb, APC, BRCA1/2, Nf2, VHL, Pten, E-cadherin) Viable TS gene knockouts often develop distinct tumors (e.g. lymphomas and sarcomas in p53 knockouts) Conventional TS gene knockouts do not recapitulate sporadic tumor formation in humans: No interaction with normal cells No control over timing: Mutations present in germline Mutations might trigger compensatory pathways

52 A third choice: reproducing sporadic human cancer in mice by genetical engineering sporadic cancer controlled switching conventional transgenic/knockout controlled switching of multiple mutations Epithelial cell with 1 mutation Mutagen/radiation Epithelial cell with 2 mutations Epithelial cell with 3 or more mutations (invasive) Controlled switching

53 How can we model sporadic cancer in mice?

54

55

56

57 Recombinase-mediated gene switching loxp loxp TS protein 1 pa 2 * oncoprotein 2 + Cre 3 pa + Cre * TS protein * 1 2 oncoprotein pa Jonkers and Berns, Nat Rev Cancer 2002

58 Induction of tumors by tissue-specific Cre expression Lung tumors: Intratracheal Adeno-Cre Mesotheliomas: Intrathoracic Adeno-Cre Mammary tumors: Epithelium-specific K14-Cre

59 Conditional mouse models of breast cancer Use P53 inactivation as a starting point P53 is involved in many types of human tumors, including breast cancer Conventional p53 knockouts develop lymphomas/sarcomas rather than carcinomas Produce conditional p53 knockouts in which p53 is inactivated in epithelial tissues

60 Conditional mouse models of breast cancer Keratin14-cre (K14cre) transgenic mice Conditional p53 (p53 F ) knockout mice

61 Tumor formation in K14cre;p53 F/F females % tumorfree 100 mammary tumors skin tumors other tumors K14cre; p53 F/F (n =24 ) latency (days)

62 Keratin 14-Cre, Brca1 flox/flox, p53 flox/flox (KB1P) mouse model Brca1 ko embryonic lethal Tissue-specific inactivation Keratin14-Cre Brca1 F loxp p53 F Liu et al. PNAS 2007

63 Keratin 14-Cre, Brca1 flox/flox, p53 flox/flox (KB1P) mouse model Brca1 ko embryonic lethal Tissue-specific inactivation Keratin14-Cre Brca1 F loxp p53 F Liu et al. PNAS 2007

64 Keratin 14-Cre, Brca1 flox/flox, p53 flox/flox (KB1P) mouse model Brca1 ko embryonic lethal Tissue-specific inactivation Keratin14-Cre X Brca1 F loxp X p53 F loxp Liu et al. PNAS 2007

65 Synergistic tumor suppressor activity of BRCA1 and p mammary tumors skin tumors other tumors % tumorfree 50 K14cre;p53 F/F K14cre;Brca1 F/F ;p53 F/F latency (days)

66 BRCA1 mouse mammary tumors resemble human BRCA1-associated breast cancer High grade solid carcinoma (IDC) Undifferentiated Pushing margins ER- PR- and HER2-negative Basal-like Genomic instability K14cre;p53 F/F K14cre;Brca1 F/F ;p53 F/F Log2(ratio) Log2(ratio)

67 E-cadherin loss results in a switch from invasive ductal carcinoma to invasive lobular carcinoma K14cre;p53 F/F Ductal carcinoma K14cre;Ecad F/F ;p53 F/F Lobular carcinoma CK14 E-cadherin

68 E-cadherin loss induces tumor metastasis Muscle Axillary LN GI tract H&E CK8

69 How can we visualize metastatic tumors? Luciferase-based in vivo imaging Makes it possible to follow tumor growth and therapy response in time Produce luciferase in tumor cells Luciferine + ATP + O 2 Oxyluciferine + AMP + Light Luciferase Inject luciferase transgenic mice with luciferin Measure photon-emission with an ultra-sensitive CCD camera

70 A conditional luciferase reporter (LucRep) mouse for in vivo imaging Actin STOP luc Creexpression in germline Actin luc Light

71 Imaging of Cre-induced tumors in LucRep mice Conditional GEM model of human cancer LucRep mouse Compound conditional mouse Cre expression (Cre-ERT, Adeno-Cre or Cre transgene) Bioluminescent tumor

72 E-cadherin loss induces tumor metastasis K14cre;Ecad F/F ;p53 F/F

73 Take home messages Genetically engineered mouse models are a useful complementary tool to study various diseases, including cancer The CRISPR/Cas9 technology has revolutionized genetic engineering

74 Acknowledgements Rottenberg group Marco Barazas Joana Barbosa Sohvi Blatter Carmen Disler Natalia Domanitskaia Alexandra Duarte Paola Francica Nora Gerhards Ewa Gogola Charlotte Guyader Denise Howald Daria Iellamo Janneke Jaspers Asli Kucukosmanoglu Martin Liptay Merve Mutlu Myriam Siffert Guotai Xu Collaborators NKI Stefano Annunziato Piet Borst Julian de Ruiter Jos Jonkers Marieke van de Ven Hans Clevers Norman Sachs