Modelos de enfermedades humanas en C.elegans

1. Modelos
de
enfermedades
humanas
en
C.
elegans
Julián Cerón Madrigal!
Laboratori de Genètica Molecular, IDIBELL, Barcelona!
Métodos
innovadores
para
reemplazar
la
experimentación
animal

2. CRISPR
RETINOSIS
PIGMENTARIA
CANCER

3. 1961
Discovery of
the genetic code:
every triplet of nucleotides
codes for one aminoacid

4. Le0er
from
Brenner
to
Perutz,
1963
It
is
now
widely
realized
that
nearly
all
the
“classical”
problems
of
molecular
biology
have
been
solved.
I
would
like
to
tame
a
small
metazoan
organism
to
study
development
directly.

6. First C. elegans publication, 1974

7. -­‐
959
somaBc
cells
-­‐
302
neurons
-­‐
Hermaphrodites
-­‐
Easy
to
the
grown
in
the
lab
-­‐
Strains
can
be
frozen

8. C. elegans is small (0.25 to 1 mm long)
DissecJng
microscope

9. C. elegans
life cycle

10. www.wormbase.org
www.wormbook.org
Easy (and cheap) to cultivate
Efficient for genetic analysis
Invariable lineage
Transparent

11. Apoptosis
C.
elegans
has
exactly
959
somaJc
cell

12. The apoptotic pathway is conserved

13. Three Nobel Prizes
2002 2006 2008

14. 1994
Osamu
Shimomura
MarJn
Chalfie

16. RNAi by feeding in C. elegans

17. Aging
research

19. Tools
and
techniques
1974 1998
2011 2015

20. CRISPR
RETINOSIS
PIGMENTARIA
CANCER

22. Convenience of C. elegans for CRISPR

23. CRISPR in C. elegans research
- Produce mutations: point mutations or deletions
- Produce endogenous reporters

24. The use of reporters in model systems

25. EXTRACHROMOSOMAL
(high copy)
3XFLAG tag
IF anti-FLAG
ENDOGENOUS
REPORTER

28. Previous
methods
for
generaJng
endogenous
reporters
Paix
et
al.,
2016
FP
inserJon
with
PCR
product
repair
templates
(75%
efficiency
in
gtbp-­‐1)
Dickinson et al., 2015
FP insertion with repair template plasmid
(12.5% efficiency in his-72)

29. In our hands: 1 positive after 72 injections and 366 worms genotyped
2016

30. Jeremy Vicencio
26 %
41 %

31. Vicencio et al , 2019

32. Diverse nucleases with distinct PAM sequences,
most of them not commercially available as protein

33. Cas9 vs Cpf1 (Cas12a)

35. New CRISPR systems in C. elegans to
evaluate:
- Toxicity
- Specificity
- Efficacy

36. Trends in Cell Biology DOI: (10.1016/j.tcb.2019.08.004)
Copyright © 2019 Elsevier Ltd Terms and Conditions

37. CRISPR
RETINOSIS PIGMENTARIA
CANCER

40. Prevalence:
1:4000
people
15000-­‐16000
affected
in
Spain
Retinitis Pigmentosa
No
cure
for
it
Mechanism
not
unveiled
≈50%
without
geneBc
diagnosBc
NORMAL VISION TUNEL VISION

41. 24 genes have been
associated to adRP
7 SPLICING-RELATED GENES:
PRPF3, PRPF4, PRPF6, PRPF8, PRPF31,
SNRPN-200 , RP9
Autosomal dominant Retinitis Pigmentosa and splicing
Autosomal dominant form: 30-40%

42. A C. elegans model for Retinitis Pigmentosa
Rubio-Peña et al, 2015

43. prp-­‐8(cer14[R2302del])
III
prp-­‐8(cer22[R2303G])
III
snrp-­‐200(cer23[V676L])
II
snrp-­‐200(cer24[S1080L])
II
Karinna Rubio-Peña
(PRPF8)
(SNRNP200)
Splicing-related Retinitis Pigmentosa mutations in C. elegans

44. CRISPR to mimic human mutations in worms
1-­‐
Study
mechanisms
of
the
disease
2-­‐RNAi
screens
to
study
funcJonal
interacJons
of
the
mutated
protein,
and
uncover
modifiers
of
the
disease
3-­‐
Drug
screens

45. Splicing-related Retinitis Pigmentosa mutations in C. elegans
Type of mutation Allele Description Phenotype
p rp -8
point mutation cer22 R2310/2303G No obvious phenotype
indel cer14 H2309/2302del pSte, pLva, pEmb
s n rp -200
point mutation cer23 V683/676L pLva, pEmb, pSte, pMlt
point mutation cer24 S1087/1080L No obvious phenotype

46. Compounds
DMSO
933
compounds
screened
in
prp-­‐8(cer14)
and
WT
4
drugs
validated
on
agar
Doxycycline
Flutamide
Dequalinium Cl
Dronedarone
Wild
Type
adRP
mutants
Mild
effect
Strong
effect
Some FDA-approved drugs can be harmful for adRP patients

47. prp-8 or snrp-200 mutants Rescue??
Drug
amenable to
high-throughput
screening
Drug screen

48. Functional replacement of RP genes
Human
Prpf3
683
aa,
16
Exons
C.
elegans
prp-­‐3
621
aa,
5
Exons

49. Kiser
et
al.
2018
Screen for modifiers of the disease

50. Validation of hits from the RNAi screen
N
=
2

51. CRISPR
RETINOSIS
PIGMENTARIA
CANCER

52. Cancer-related mutations mimicked in C. elegans
Avatar
Worms
(Quesada
et
al.
2011)
SF3B1
is
mutated
in:
20-­‐28%
myelodisplasic
syndromes
(MDSs).
5-­‐18%
chronic
lymphocyBc
leukemias
(CLL).
15-­‐20%
uveal
melanomas,
5.6%
breast
cancer

53. C. elegans as a pre-clinical model to identify selective treatments
Normal
worms
sHb-­‐1
Mutant
worms
sHb-­‐1*
Tumor
cells
SF3B1*
Normal
cells
SF3B1

54. 5’-CTCGAATTGCTTAAAGCTCACAAGAAAAGTATTCGAAGAGCTGCAATTAATAC/ATTTGGATTTATTG
S1090
A1095
I1096
F1101
WT
MUTANT
A1090
T1095
V1096
Y1101
5’-CTCGAATTGCTTAAAGCTCACAAGAAAgcgATcaGAcGAGCaaCtgTgAAcAC/tTTcGGtTacATTG
Humanizing sftb-1 to make it sensitive to Pladienolide

55. Humanized sftb-1 is sensitive to Pladienolide

56. Resistance to cisplatin-based chemotherapy
ü
EffecBve
treatment
for
many
cancer
types
×
Some
tumors
are
resistant
to
cisplaBn
theraphies
(intrinsically
or
acquired)
×
Side-­‐effects
CISPLATIN

57. Identification of genes involved in cisplatin resistance/sensitivity
Functional validation of candidate genes to confirm resistance/sensitivity
C. elegans and chemotherapy

58. C.
elegans
to
validate
targets
About
100
candidate
genes
to
influence
cisplaJn
response
in
the
region
9q32-­‐q33.1.
Which
of
those
genes
are
implicated
in
the
cisplaBn
resistance
acquisiBon?
Dr
Alberto
Villanueva
(Piulats
et
al,
Clinical
Cancer
Research
2018)

59. García-Rodríguez et al. Dis. Model. Mech. 2018
C. elegans to indetify new genes involved in chemoresistance

60. Modified
from
Galluzzi
et
al.,
2012)
Cisplatin: molecular mechanism of action
ROS
Adapted
from
Waissbluth
and
Daniel,
2013

61. F27C1.2/CTR1A
C.
elegans
mutants
display
resistance
to
cisplaJn

62. Cisplatin transporters in human, worms and flies

65. Biomedical
Research

66. C.
elegans,
a
pluricellular
model
that
can
fill
the
gap
between
in
vitro
and
translaBonal
studies
In
vitro
studies
Preclinical
research
Clinic
Basic
research
Pre-­‐
preclinical
research

69.
Xènia
Serrat
Carmen
Marinez
David
Brena
Dmytro
Kukthar
Jeremy
Vicencio
Isabel
García