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