Genetically modulated substrate reduction therapy

(1)

GENETICALLY

MODULATED

SUBSTRATE REDUCTION

THERAPY

Maria Francisca Coutinho

Lysosomal Storage Disorders Group Research & Development Unit,

Department of Human Genetics, INSA

11th_{B4B WorkShop}

(2)

enzyme replacement

11th_{B4B WorkShop} 29th_March_-1st_April_{, 2017}

THE

ENZYME

AS

A

DRUG

?

(3)



1996



Norman Radin

THE

ENZYME

AS

A

DRUG

?

(4)

substrate reduction

enzyme replacement

(5)

(6)

SUBSTRATE

REDUCTION

THERAPY

(SRT)

Pathology

Approved SRT

Gaucher

Zavesca

®

(Miglustat; Actelion)

Cerdelga

®

(Eliglustat tartrate; Genzyme)

Niemann-Pick C

_Zavesca

®

(Miglustat; Actelion)

(7)

SUBSTRATE

REDUCTION

THERAPY

(SRT)

(8)



Spanish group

 Target:

GCS

gSRT

PROOF

OF

PRINCIPLE

- GD

11th_{B4B WorkShop} 29th_March_-1st_April_{, 2017} Diaz-Font et al., (2006) Glucosylceramide synthase

(9)



Spanish group

 Target:

GCS

gSRT

PROOF

OF

PRINCIPLE

- GD

11th_{B4B WorkShop} 29th_March_-1st_April_{, 2017} Diaz-Font et al., (2006) Glucosylceramide synthase

(10)

 Target:

GCS

 Test in:

HeLa cells

gSRT

PROOF

OF

PRINCIPLE

- GD

http://www.ensembl.org/Homo_sapiens/Transcript/Summary?db=core;g=ENSG00000133937;r=14:94768216-94770230;t=ENST00000238558 (adapted)

(11)

 Target:

GCS

 Test in:

HeLa cells

 Compound(s):

si/shRNAs

4

11th_{B4B WorkShop} 29th_March_-1st_April_{, 2017} Diaz-Font et al., (2006)

gSRT

PROOF

OF

PRINCIPLE

- GD

(12)

 Target:

GCS

 Test in:

HeLa cells

 Compound(s):

si/shRNAs

Diaz-Font et al., (2006)

(13)

 Target:

GCS

 Test in:

HeLa cells

 Compound(s):

si/shRNAs

dose-dependent effect

gSRT

PROOF

OF

PRINCIPLE

- GD

(14)

 Target:

GCS

 Test in:

HeLa cells

 Compound(s):

si/shRNAs

dose-dependent effect

gSRT

PROOF

OF

PRINCIPLE

- GD

(15)

 Target:

GCS

 Test in:

HeLa cells

 Compound(s):

si/shRNAs

effect on GCS activity (72h)

gSRT

PROOF

OF

PRINCIPLE

- GD

(16)

 Target:

GCS

 Test in:

HeLa cells

 Compound(s):

si/shRNAs

effect on glucosylceramide formation (TLC)

gSRT

PROOF

OF

PRINCIPLE

- GD

(17)

 Target:

GCS

 Test in:

HeLa cells

 Compound(s):

si/shRNAs

effect on glucosylceramide formation (TLC)

gSRT

PROOF

OF

PRINCIPLE

- GD

11th_{B4B WorkShop} 29th_March_-1st_April_{, 2017} Diaz-Font et al., (2006) 20-fold

(18)

 Target:

GCS

 Test in:

HeLa cells

 Compound(s):

si/shRNAs

coded in a DNA expression vector stable transfected cells

permanent re

d

uction of expression

gSRT

PROOF

OF

PRINCIPLE

- GD

(19)

 Target:

GCS

 Test in:

HeLa cells

 Compound(s):

si/shRNAs

2 gSRT

PROOF

OF

PRINCIPLE

- GD

(20)

 Target:

GCS

 Test in:

HeLa cells

 Compound(s):

si/shRNAs

effect on

GCS activity

gSRT

PROOF

OF

PRINCIPLE

- GD

(21)

 Target:

GCS

 Test in:

HeLa cells

 Compound(s):

si/shRNAs

effect on

GCS activity

gSRT

PROOF

OF

PRINCIPLE

- GD

(22)

SUMMARY

si/shRNAs

gSRT

PROOF

OF

PRINCIPLE

- GD

GCS

mRNA l

e

v

els

GCS

enzyme ac

t

i

vity

glucosylceramide

s

y

n

thesis

(23)

Gaucher

Fabry

Niemann-Pick

type C

Pompe

Mucopolysaccharidosis

type I

Sialidosis

Galactosialidosis

MPS IIIA

MPS II

MPS IIIB

MPS IIIC

Krabbe

Mucolipidosis

type II

Tay–Sachs

Metachromatic

leukodystrophy

Multiple sulfatase

deficiency

ML III

Farber

Sandhoff

MPS IVA

MPS IVB

Fucosidosis

Alpha-

mannosidosis

Beta-mannosidosis

Pycnodysostosis

Danon

Wolman

(24)



Chronic



Progressive



Large spectrum of severity

& symptoms

MPS III

(= Sanfilippo Syndrome)

A, B, C and D

(25)

A

VAILABLE

T

HERAPIES



Enzyme Replacement Therapy



Only available for 4 MPSs!



No effect on neurological symptoms!

(26)

A

VAILABLE

T

HERAPIES



Enzyme Replacement Therapy



Only available for 4 MPSs!



No effect on neurological symptoms!

… antibodies!

Perfect Target

for

Substrate R

e

du

ction

(27)



Polish group

 Targets: XYLT1, XYLT2, GALTI, GALTII

(28)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES



Polish group

(29)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES



Polish group

(30)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES



Polish group

 Test in: MPS IIIA fibroblasts

(31)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES



Polish group

 Compounds: siRNAs

8 ≠

(32)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES



Polish group

protein levels

(33)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES



Polish group

GAG synthesis

(34)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES

Dziedzic et al., (2010)

SUMMARY

“

early stages”

GAGs biosynthesis

genes

(35)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES

Would the simultaneous use of 2 species of siRNAs be more

effective than the use of single siRNAs?

(36)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES

Would the simultaneous use of 2 species of siRNAs be more

effective than the use of single siRNAs?

(37)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES

Would the simultaneous use of 2 species of siRNAs be more

effective than the use of single siRNAs?

not statistically significant

(38)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES



Would the simultaneous use of 2 species of siRNAs be more

effective than the use of single siRNAs?

Potential benefit:

doubtful

Cost-benefit?

Side-effects?

(39)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES 11th_{B4B WorkShop} 29th_March_-1st_April_{, 2017} 

What about…



cytotoxic effects?...



efficacy gSRT vs ERT?...



effect of combiatory treatment?...

Chmielarz et al., (2012) MPS I MPS IIIA and MPS IIIB

(40)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES 11th_{B4B WorkShop} 29th_March_-1st_April_{, 2017}



Cytotoxic effects?...

(cell survival %)

Chmielarz et al., (2012) 24h 48h

(41)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES



Efficacy gSRT vs ERT

and

effect of combiatory

treatment?...

Chmielarz et al., (2012) MPS I – cell line 1 MPS I – cell line 2

(42)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES



Efficacy gSRT vs ERT

and

effect of combiatory

treatment?...

(43)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES



Efficacy gSRT vs ERT

and

effect of combiatory

treatment?...

(44)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES 11th_{B4B WorkShop} 29th_March_-1st_April_{, 2017}



Cytotoxic effects?...

No!

However,

some cytotoxic effects could occur in particular cell lines



Efficacy gSRT vs ERT

and

effect of combiatory

treatment?...

Depends on specific features of each cell line

Thus,

the combined therapy might potentially result in various

effects in different patients

(45)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES



Australian group

 Targets: EXTL2 and EXTL3

(46)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES



Australian group

 Test in: 293T cells; MPS I and MPS IIIA fibroblasts

http://www.sciencellonline.com/products-services/primary-cells/human-dermal-fibroblasts-adult.html

Kaidonis et al., (2012)

https://upload.wikimedia.org/wikipedia/commons/8/85/HEK_29 3_cells_grown_in_tissue_culture_medium.jpg

(47)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES



Australian group

 Compounds: shRNAs

(overexpression)

(48)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES



Australian group

(endogenous)

(49)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES



Australian group

GAG synthesis

(50)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES



Australian group

GAG synthesis

(51)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES



Spanish group

(52)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES

Spanish group

 Test in: MPS IIIC fibroblasts 2

(53)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES

Spanish group

 Test in: MPS IIIC fibroblasts 2

(54)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES

Spanish group

 Test in: MPS IIIC fibroblasts

(55)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES

Spanish group

(56)

gSRT

FOR

OTHER

LSD

S

-

M

UCO

P

OLY

S

ACCHARIDOSES

Spanish group

Canals et al., (2015)

3 days 3 days

(57)

SUMMARY

si/shRNAs

mRNA l

e

v

els

enzyme ac

t

i

vity

GAGs

s

y

n

thesis

R

e

v

e

rse

the

cellular

phenotype

!!!

(58)

gSRT

FOR

OTHER

LSD

S

-

POMPE



American group

 Target:

muscle specific glycogen synthase (Gys1)

 Test in:

Pompe mice

(59)

gSRT

FOR

OTHER

LSD

S

-

POMPE



American group

 Target:

muscle specific glycogen synthase (Gys1)

 Test in:

Pompe mice

 Compound(s):

GS-PPMO

phosphorodiamidate morpholino oligonucleotide

conjugated to a cell penetrating peptide

therapy for Duchene muscular dystrophy

(60)

gSRT

FOR

OTHER

LSD

S

-

POMPE



American group

 Target:

muscle specific glycogen synthase (Gys1)

 Test in:

Pompe mice

 Compound(s):

GS-PPMO

phosphorodiamidate morpholino oligonucleotide

conjugated to a cell penetrating peptide

http://www.ensembl.org/Mus_musculus/Transcript/Summary?db=core;g=ENSMUSG00000003865;r=7:45434844-45456619;t=ENSMUST00000003964 (adapted) Cys1 Exon skipping PTC



(61)

gSRT

FOR

OTHER

LSD

S

-

POMPE

American group

 Target:

muscle specific glycogen synthase (Gys1)

 Test in:

Pompe mice

 Compound(s):

GS-PPMO

tail vein injection every 2 weeks t= 12 weeks

(62)

gSRT

FOR

OTHER

LSD

S

-

POMPE

American group

 Target:

muscle specific glycogen synthase (Gys1)

 Test in:

Pompe mice

 Compound(s):

GS-PPMO

(63)

gSRT

FOR

OTHER

LSD

S

-

POMPE

American group

 Target:

muscle specific glycogen synthase (Gys1)

 Test in:

Pompe mice

 Compound(s):

GS-PPMO

skeletal muscles

(64)

gSRT

FOR

OTHER

LSD

S

-

POMPE

American group

 Target:

muscle specific glycogen synthase (Gys1)

 Test in:

Pompe mice

 Compound(s):

GS-PPMO

cardiac muscles

(65)

gSRT

FOR

OTHER

LSD

S

-

POMPE

American group

 Target:

muscle specific glycogen synthase (Gys1)

 Test in:

Pompe mice

 Compound(s):

GS-PPMO

tissue-specific effect

(66)

gSRT

FOR

OTHER

LSD

S

-

POMPE

American group

 Target:

muscle specific glycogen synthase (Gys1)

 Test in:

Pompe mice

 Compound(s):

GS-PPMO

lysosomal glycogen accumulation

(67)

SUMMARY

Target protein l

e

v

els

lglycogen

l

e

v

els

Tissue-specific effect

(68)

A LOOK FORWARD…



Vector design & si/shRNA encapsulation

 bioavailability of siRNAs;  protection from degradation  control of  circulation time  release rate Lipid nanoparticles? siRNA-peptide conjugates? Cell-penetrating peptides (CPP) 11th_{B4B WorkShop} 29th_March_-1st_April_{, 2017}

(69)

A LOOK FORWARD…



Vector design & si/shRNA encapsulation

 bioavailability of siRNAs;  protection from degradation  control of

 circulation time  release rate



Genotoxic evaluation of the

si/shRNA+nanocarrier formulation(s)

 internalization  cell viability  genotoxic potential 11th_{B4B WorkShop} 29th_March_-1st_April_{, 2017}