Mucopolysaccharidosis and
adenovectors (CAV)
Terapia genica
18/4/2012
Anna Borroso
Gianmarco Rinaldi
Petra Tomassini
Silvia Santopolo
The mucopolysaccharidoses (MPSs) are lysosomal storage disorders
caused by the accumulation of glycosaminoglycans in the lysosomes that
leads to a cascade of multisystemic disease manifestations. The disease is
caused by mutations in one of the enzymes that catalyze GAGs
degradation.
The most severe forms of MPSs involve progressive degeneration of the
Central Nervous System with consequent cognitive impairment. Current
therapies are targeted to the replacement of the defective enzyme, but the
impossibility of therapeutic molecules or vectors to cross the Blood Brain
Barrier limits the treatment to the non- neural symptoms.
The aim of new emerging strategies is the reduction of the substrate
production, rather than promote its degradation. According with this
strategy, we propose to inhibit the gene expression of the enzymes involved
in the first steps of GAG biosynthesis using shRNAs expressed by an
helper-dependent canine adenoviral vector. Our aim is to effectively treat
cognitive impairment, which is still now uncured.
Abstract
- Lysosomal storage disorder
- Deficiency of one of the enzyme
involved in degradation of GAGs.
- Seven distinct clinical types(I-II-III-IVVI-VII-IX)
- Autosomal recessive disorder (the
exception is MPS II that is X-linked)
- Affect multiple organ systems and lead
to organ failure
- Cognitive impairment
- Reduced life expectancy
Mucopolysaccharidosis
Site of the enzyme deficiencies
- autosomal recessive
- 1:100 000 frequence
- 3 type:
- Hurler Syndrome
- Hurler-Scheie Syndrome
- Scheie Syndrome
- deficient activity of α-L-iduronidase
(IDUA) (chromosome 4)
- more than 100 different mutations
MPS I
Strategy
Degradation
of stored
GAGs via
restoration
of enzyme
activity
Inibition of new
GAGs synthesis
(substrate
reduction
therapy)
Mechanism
Limits
ERT, Enzyme replacement
Therapy
CNS access across the
Blood Brain Barrier;
Repeated
administration.
HSCT, Hematopoietic Stem Cell
Treatment
CNS access across the
BBB
Gene therapy ,
Numerous eterogeneus different
mutantions across the entire
gene: it needs to be entirely
substitueted
Immunogenicity of
IDUA;
Diffusion of the vector
into the brain;
Inhibitors of enzymes involved
in GAGs biosynthesis (Genistein)
Side Effects (e.g.
decreased male
fertility)
Gene Theray with siRNA:
Inhibition of the expression of
enzymes involved in GAG
biosynthesis
Existing Therapies
Delivery
Therapeutic strategy to recover
cognitive impairment
•
•
Reduction of GAG
accumulation throught
inhibition of their syntesis
use of siRNAs
use of Canine Adenoviral Vector
- low immunogenicity
- efficently trasduce neurons
•
Objectives
How to design the shRNAs
3rd generation
CAV-2 multi-shRNA-GFP
CAV-2 multi-shRNA-GFP
primary cultures of
neurons from
wt mice
- Test cell vitality
- Test vector presence: light microscopy
observation of GFP fluorescence
- siRNAs expression: Northern Blot
- mRNAs target levels: Real time PCR
- Target proteins levels: Western Blot
- Reduction of GAG biosynthesis:
measurement of incorporation of 35S
from a radioactive sodium sulfate into PG
- Level of GAG: Blyscan Assay
- siRNAs off-target effects: Microarray
Experimental plan
CAV-2 multi-shRNA-GFP
primary cultures of
neurons from
MPS I mice
- Test cell vitality
- Test vector presence: light microscopy
observation of GFP fluorescence
- siRNAs expression: Northern Blot
- mRNAs target levels: Real time PCR
- Target proteins levels: Western Blot
- Reduction of GAG biosynthesis:
measurement of incorporation of 35S
from a radioactive sodium sulfate into PG
- Level of GAG: Blyscan Assay
- siRNAs off-target effects: Microarray
- Reduction of the number of lysosomal
inclusions: light microscopy
Experimental plan
CAV-2 multi-shRNA-GFP
MPS I newborn mice
- Test vector diffusion: GFP fluorescence
observation in brain sections
- Target mRNAs levels: Real Time PCR
- Target proteins levels: Western Blot
- Reduction of GAG storage: light
microscopy observation of number and
aspect of lysosomal inclusions
- Absence of desease synthomps: cognitive
test
- Eventual IFN siRNAs response: IFN target
genes levels of expression
Experimental plan
Necessity of an early
treatment in newborn
patients
Future human trial
Pitfalls and solutions
Necessity of a
prenatal diagnosis
Necessity of a long-therm
expression of the shRNA
(e.g.: hybrid vector able
to integrate in the host
genome)
MPS I mice (IDUA-/-): provided by Dr. E. F. Neufeld, USA
Blyscan Glycosaminoglycan Kit: 120 Assays 478 €
Kit Microarray: 500€
Anticorpi vari: 500€
Strumentazioni e varie: 15 000€
Materials and costs