La genetica nel Parkinson:
attualità e prospettive
Alessandra Renieri
Medical Genetics, University of Siena
& Azienda Ospedaliera Universitaria Senese
Pathways underlying PD pathogenesis
Hum Mol Genet. 2007;16(R2):R183-R194. doi:10.1093/hmg/ddm159
Medical Genetics of Siena
Case 1 The patient is a 53-year-old female who
developed PD symptoms at the age of 47 years.
The disease started with bradykinesia, severe
dyskinesias and rigidity.
Currently, psychiatric disturbances are not present
and the patient is treated with levodopa.
Her parents are still alive, are 78 and 75 years
old, and a recent neurological examination did not
show any sign of PD. DNA from the parents is not
available for genetic testing.
The proband has a healthy 16-year-old son.
Cons. 2007
LRRK2 mutation c.G2019S
Squillaro et al J Hum Genet 2007
Science 06 Apr 2018 Vol. 360, Issue 6384, pp. 36-37
DOI: 10.1126/science.aar5683
Two LRRK2 inhibitors are currently
being evaluated to treat Parkinson’s in
two Phase 1 trials.
The experimental therapies, called
DNL-201 and DNL-151, are being
developed by Denali Therapeutics.
So far, DNL201 has stopped an
average 90 percent of LRRK2 kinase
activity at its highest concentration.
When the drug’s levels dropped to the
lowest concentration, it still inhibited on
average 50 percent of such activity.
60:40 M:F in sporadic cases
versus
40:60 M:F in LRRK2 mutated cases
MODELLO A SOGLIA- EFFETTO DEL SESSO
Predisposizione
Num
ero
di in
div
idui
MASCHI
Predisposizione
Num
ero
di in
div
idui
FEMMINE Femmina affetta
+ predisposizione
MAGGIORE rischio di ricorrenza
Es. stenosi del piloro
M:F 5:1
Cellular pathogenetic processes
Medical Genetics of Siena
Case 2
Diagnosis of Parkinson’s disease at 50 y:
• disturbances in movement planning
• rigidity
• dyskinesias
• postural instability
• behavioral disorders
• hallucinations
• phenomena on-off
GBA mutation c.T882G, p.H294Q
Parkinson’s disease diagnosis at 30 y
Cons. 2017
BACKGROUND
The efficacy of current therapies in Parkinson disease
is time-bound
L-DOPA
treatment
other
treatments
L-DOPA
treatment
other
treatments
disease
progression
TARGET
TACGGCA
LRRK2 gene
To correct specifically the patient LRRK2 mutation, or mutation in other causative genes
(Parkin, GBA, SNCA, VPS35, PINK1, DJ-1, DNAJC6 etc) of Parkinson Disease
c. G6055A
p.G2019S
STRATEGY
Mouse Model
Design and produce an AAV viruses to
precisely correct the mutations inside cells
using CRISPR/Cas9 technology
Test the the efficacy in vitro (iPSCs)
and in mouse model
Longitudinal patient analysis to identify the exact clinical timing for drug delivery for maximising the efficacy
INNOVATIONS
A) Technical Innovation B) Clinical Innovation
- The kind of
Cas9 and
mutations
thereof
- The system for
patient monitoring
- The protocol
for patient delivery
Procedures for patenting of the above innovation is ongoing at University of Siena
-The kind of viral
vector
- The target system to
Neurons
It is part of the Bacterial adaptive immune system which detects and recognizes
foreign DNA and cleaves it.
1- CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci
2- Cas (CRISPR-associated) proteins can target and cleave invading DNA in a
specific sequence
A CRISPR locus is composed by a series of repeats interspaced by spacer sequences acquired
from invading genomes
CRISP-Cas9 technology
A new gene editing tool
CRISPR system can be reconstituted in vitro
Genome specific
gRNA target
sequence
Genomic DNA
Cas9 Nuclease
KNOCK-OUT EVENTS PRECISE GENE EDITING
Target recognition through base pairing between gRNA and the genomic sequence.
Cutting process through a double-stranded blunt-end break precisely 3 nucleotides
from PAM sequence and this triggers endogenous DNA repair mechanisms.
THE CRISPR SYSTEM
has been employed since 2013 as a
simple and reliable technique for
editing, in living cells, the genomes of
mammals and other organisms
An average of one clustered
regularly interspaced short
palindromic repeat (CRISPR) Cas9
paper published every 4 h
in 2017
Moving fast into clinical practice
AAV2 vector containing
human RPE65 cDNA
subretinal injection
oligonucleotide
intrathecal administration
TIME TABLE
0 12 24 30
Vector and Virus Preparation
iPSCs Cellular Model
Mouse Model
Months
we are here
Plasmids Construction
sgRNA Plasmid
mCherry Reporter
sgRNA Donor
DNA
Cas9 Plasmid
Self-Cleaving
Cas9
CMV
Promoter
Self-Cleaving
Cas9
Cas9
AAV
sgRNA
AAV
Cas9
AAV Production and Infection
sgRNA plasmid Cas9 plasmid AAV
sgRNA AAV
Cas9
sgRNA design on LRRK2
c.6055G>A (p.Gly2019Ser)
sgRNA design on LRRK2
sgRNA design on PARK2
c.823C>T (p.Arg275Thr)
sgRNA design on GBA
c.1226A>G (p.Asp409Ser)
spCas9 Canonical NGG spCas9 Non-Canonical NAG
mCherry GFP merge
Plasmids are efficently transfected in Fibroblasts
by Lipofectamine : Immunofluorescence analysis
non corrected cells corrected cells
Efficient editing (89%) NGS analysis in fibroblasts
AAVs production
Helper Plasmids
Co-transfection in Packaging
cells
DAPI/GFP/ Tubulin Neurons Infected by AAV9 (48 h)
iPSC-derived Neurons can be efficiently
infected by AAV: Immunofluorescence analysis
AAVs
Virus production
Gene correction
iPSCs
AAV9
iPSCs
Fibroblasts
In vitro analysis Lymphoblasts
Neurons
Medical Genetics of Siena