1. No category

Malattia
Gene mutato
Distrofia muscolare di Duchenne
(DMD)
Distrofina
Distrofia muscolare di Becker
(BMD)
Distrofina
Distrofia muscolare di EmeryDreifuss
Emerina, lamina A o lamina C
Distrofia dei cingoli (LGMD)
Piu' di 15 geni diversi
autosomiche dominanti
LGMD 1A: miotilina
LGMD 1B: lamina A/C
LGMD 1C: caveolina 3
ed altre
autosomiche recessive
LGMD 2A: calpaina-3
LGMD 2B: disferlina
LGMD 2C: γ-sarcoglicano
LGMD 2D: α-sarcoglicano
LGMD 2E: β-sarcoglicano
LGMD 2F: δ-sarcoglicano
ed altre
Distrofia facio-scapolo-omerale o di Non noto
Lnadouzy-Dejerine (FSHD)
Distrofia miotonica o malattia di
Steinert (MMD)
DMPK (DM1) e ZNF9 (DM2)
Distrofia oculo-faringea (OPMD)
Poly(A)-binding protein nuclear 1 (PABPN1)
Distrofia muscolare distale (DD)
Almeno 8 geni diversi (disferlina, titina, desmina ed altri)
Distrofia muscolare congenita
(CMD)
Geni diversi (Laminina α2 – merosina, fukutina, collagene di tipo
VI, integrina a7, ed altri)
Challenges to gene therapy for DMD
La DMD ha un decorso ingravescente e
devastante (esaurimento delle cellule satelliti).
Alla nascita, i bambini maschi affetti sembrano
normali, ed i primi sintomi insorgono tra i 3 ed
i 5 anni di vita sotto forma di blanda debolezza
muscolare, che si manifesta con la difficolta'
nel salire le scale, alzarsi nella posizione
seduta o con l'incespicare di frequente. Con il
passare del tempo, la muscolatura si
indebolisce progressivamente.
Solitamente entro i 10 anni di vita gli individui
affetti sono costretti sulla sedia a rotelle, e
molti decedono entro il 20 anno di eta'.
Gene therapy requires delivery of a new gene to the vast majority of muscles
in the body - a daunting challenge, since muscle tissues makes up >40% of
body mass
The dystrophyn mRNA is 14 kb in size. A delivery vector must be identify that
can carry this expression cassette; alternatively, truncated version of the gene
can be used
Non esistono attualmente terapie per la
malatia, se non quelle di supporto.
Oltre al muscolo scheletrico, i pazienti con
DMD mostrano un interessamento piu' o meno
marcato del cuore che spesso evolve in una
forma franca di cardiomiopatia dilatativa.
Muscle transduction must not trigger toxic or immunological reactions that are
harmful to the patient or that lead to further muscle damage
Vectors for muscle gene therapy
• Ad “gutless”: Ad vectors have relative large cloning capacity,
can be grown at high titers and display relative efficient infection
of muscle. However, traditional Ad vectors elicit a robust cellular
immune response
• AAV: AAV vectors are of great interest as they efficiently infect
muscle and persist for years. New serotypes available
• Plasmid DNA: displays a remarkable ability to transfer genes to
muscle, specially if coupled with high pressure injection and/or
electroporation
The first clinical trial, closed in 2006, entailed the injection of a plasmid
containing the whole dystrophin cDNA under the control of the CMV
promoter into the radialis muscle of 9 DMD/BMD patients. However,
dystrophin expression resulted too low and not homogenous.
Different strategies can be used to increased transduction efficiency,
including polymeres, ultrasounds (with microbubbles), and electroporation
INFEZIONE
PRODUTTIVA
Agenti che inducono uno
stress cellulare
AAV
Infezioni virali
Adenovirus
Herpesvirus
Papillomavirus
Taxonomy
Agenti fisici
Raggi gamma
Raggi X
UV
Family: Parvovirus
Subfamily: Parvovirinae
Genus: Dependovirus
Type: AAV 1-5
INFEZIONE
LATENTE
Metil-metan sulfonato
Mitomicina c
Cisplatino
Idrossiurea
Inibitori della
topoisomerasi
Inibitori delle proteasi
Heat shock
Agenti chimici
Ad
Replicazione del
DNA virale
Morphology
Particles are icosahedral, non-enveloped,
18-26 nm diameter, 50% protein (VP1-3) 50% DNA.
Resistant to inactivation by solvents, pH and heat.
19p13.3
19q13.2
19p13.2
19p13.1
19p12
19q12
19q13.31
19q13.32
19q13.33
19q13.41
Genome
19q13.1
19q13.42
Linear, non-segmented, ssDNA ~5 kb.
AAVs package equal amounts of (+) and (-) strands.
Produzione di
nuove particelle
virali e lisi della
cellula
AAVS1
19q13.2
19q13.3
19q13.43
19q13.4
Integrazione nel cromosoma
19q13.3
Xie et al. 2002
Therapeutic gene
Rep-Cap
Helper
plasmid
AAV
vector
Ad helper genes
RBS’
C
Rep
binding
site
(RBS)
6 hrs
CPE
293 cells
3’
5’
B
A
CaPo4 transfection
Terminal
resolution
site (trs)
5’ and 3’ Inverted Terminal
Repeats have a predicted
hammer-head like secondary
structure
During AAV replication Rep bind
the ITR and cleaves one strand
48 hrs
Freeze/Thaw
Amm sulf fractionation
rAAV
CsCl gradient
purification
Collect and dialyze fractions
Titration by QC-PCR
Assay for transgene expression
Xie et al. 2002
1. Based on a widely diffused, non pathogenic
virus
AAV
Heart
2. Vectors retain less that 10% of the viral
genome
Tibialis anterior
Ad
3. Vectors do not express viral proteins (not
inflammatory and not immunogenic); permit
long term persistence in vivo
Diaphragm
AAV-lacZ
3’
Skeletal muscle
4. Expression of the therapeutic gene can be
driven by any desirable promoter
5. High titer vector preparations can be
obtained by virion purification
6. Cells are transduced at high multiplicity of
infection; mixing of different rAAV
preparations results the simultaneous
expression of gene combinations in vivo
AAV-lacZ
Heart
wt AAV
p5
TR
p19
p46
Rep
polyA
Cap
TR
rAAV vector
Therapeutic gene
TR
Neurons
AAV-GFP
Retina
AAVLacO.14
Mre11
Merge
AAVLacO.14
Rad50
Merge
HU
TR
AAV-LacZ
polyA
Promoter
AAVLacO.14
(14 8-mers, 112 LacO repeats)
LacO (8-mer)
HU
3’
HeLa
LacR-EGFP
HU
3’
Nbs1
Merge
HU
AAVLacO.14
Untreated
(+)
Merge
(-)
rAAV foci
(+)
DNA synthesis
and/or
+/- strand
annealing
HU
(-)
Inhibition of
vector DNA
processing
Untreated
AAVLacO.14 P-S343-Nbs1
Binding to
cellular DDR
proteins
1.5
Mre11
Rad50
Nbs1
1.0
0.5
0.0
NH
H
Efficient
transduction
Parvovirus
Recettore
AAV1
Acido sialico (legami α2-3-N e α2-6N)
AAV2
Proteoglicani contenenti eparansolfati (HSPG)
Corecettori: integrina αvβ5, FGFR1,
HGF-R
Sierotipo (in ordine di
efficienza)
AAV3
Proteoglicani contenenti eparansolfati (HSPG)
Fegato
AAV8, AAV9
AAV4
Acido sialico (legami α2-3-O)
Muscolo scheletrico
AAV5
Acido sialico (legami α2-3-O e α2-3N)
Recettore del PDGF (PDGFR)
AAV1, AAV7, AAV6, AAV8,
AAV9, AAV2, AAV3
oso centrale
Sistema nervo
AAV5, AAV1, AAV4, AAV2
Acido sialico (legami α2-3-N e α2-6N)
Occhio
AAV5, AAV4, AAV1, AAV6
AAV6
http://
www.icgeb.org/
avu-corefacility.html
Organo
AAV7
Non noto
AAV8
Recettore della laminina (LamR)
Epitelio
pigmentato
Heart
Liver
Fotorecettori AAV5
AAV9
Non noto (LamR?)
Polmone
AAV5, AAV9
Parvovirus B19
Antigene P dei globuli rossi
Cuore
AAV9. AAV8
CPV (parvovirus canino)
Recettore della trasferrina
Acido sialico (acido N-glicolilneuraminico, NeuGC)
Pancreas
AAV8
Rene
AAV2
FPV (parvovirus della
panleucopenia felina)
Recettore della trasferrina
Tibialis anterior
Diaphragm
Helper Plasmids
Neurons, Muscle
The AVU produces, purifies,
titers and characterizes
research-grade recombinant
AAV vectors
Rep
Cap1
Ad (E2A, VA, E4)
Neurons, Muscle
Cap2
Muscle
Cap3
Neurons
Cap4
Lung, Heart
Cap5
Both rAAV2 or hybrid vectors pseudotyped
with capsid of serotypes from 1 to 9 are
reproducibility obtained at a sterility and purity
grade suitable for in vitro and animal
experimentation
Endothelium, Muscle
Cap6
or
Muscle
Rep
Cap7
Ad (E2A, VA, E4)
Liver, Heart
Cap8
Rep5
CapBAV
Ad (E2A, VA, E4)
http://
www.icgeb.org/
avu-corefacility.html
Angiogenesis
&Endothelial function
huVEGF-A165
huVEGF-A165 (Tet-ON)
huVEGF-A165-HA
huVEGF-A121
muVEGF-A164
muVEGF-A120
huVEGF-D full length
huVEGF-D processed
huVEGF-B
VEGF/FGF4
Angiopoietin 1
Semaphorin 3A
PlGF
PDGF-B
TRAIL
HIF-1α-VP16
Netrin1
Development
&Regeneration
Islet-1
Msx-1
Notch ICD
Cripto
c-Myc
Klf4
Oct3/4
Sox2
Cardiac
&muscle function
IGF-1
Melusin
ZASP1
Ghrelin
Cytokines
&Chemokines
RANTES
Interleukin 1β
Interleukin 1β NG
RGMa
MCP-1
MIP-1a
PET Imaging
HSV-1 TK
HSV-1 TKsl39
HIV-1 Tat-HSV-1 TK
HIV-1 Tat-HSV-1 TKSL39
HIV-1
HIV-1 Tat
HIV-1 Tat-SP
Apoptosis
Bcl-2
shRNAs
FLK1
Zacfu6/hairpin
Sh-Amb1
Sh-Amb2
ZacU6
GFP
Inflammation
IκB-α
Antibodies
ScFv-D18 (anti-Prp)
ScFv-TG (anti-TG)
Immune function
CTLA-4
ADAMTS13
Extracellular matrix
Timp-1
Pentraxin-3
Cell cycle
&Cancer
p27*
Ras
Mutated Ras
cMyc
HPV E7
Reporters
LacO binding site repeats
LacZ
GFP
GFP (Tet-ON)
HPV
E6
E7
Others
Cre-GFP
CFTR-YFP
DCN
ErB4DCF
NRG1IIIb3
Calpain1
Calpain2
PA3
PE8
Cap-YFP
Medical
Condition
Cancer
Disease
Gene
Route
Phase
Status
PI
Prostate cancer
GM-CSF
Ex vivo, intradermal
I-III
4 Open/2 Completed
Gillison ML, Drake C,
Corman JM, Curti B, Urba
WJ
Dummer R
Melanoma
B7-2, IL-12
Ex vivo, intradermal
I
1 Open
Cardiovascular
Heart failure
SERCA-2a
Intracoronary
I
2 Open
London B, Jessup M
Infectious
diseases
HIV-1 vaccination
HIV-1 gag-pro-prt
Intramuscular
I
2 Open
Clumeck N, van Lunzen J
Monogenic
diseases
Lipoprotein Lipase
deficiency
Lipoprotein Lipase S447X
Intramuscular
I-II
1 Open
Stroes E
Leber amaurosis
RPE65
Intraocular
I
2 Open
Byrne B, Jacobson S,
Maguirre A
Hemophilia B
FIX
Intramuscular/Liver
I
2 Completed/1 Under
review
Manno C, Glader B,
Nienhuis A
Cystic fibrosis
CFTR
Intranasal,
Intrapulmonary
I-II
6 Completed
Gardner P, Aitken M
Limb girdle distrophy
Sarcoglycan
Intramuscular
I
8 Open/2 Completed
Mendell J
Duchenne muscular
Dystrophy
Minidystrophin
Intramuscular
I
1 Open
Mendell J
alpha1-antitrypsin
deficiency
AAT
Intramuscular
I
2 Open
Flotte T
Parkinson disease
GAD, AADC-2, Neurturin
Intracranial
I-II
2 Open/ 1 Completed
Marks W, Verhagen L
Alzheimer disease
NGF
Intracranial
I-II
1 Completed
Bennett D
Intractable epilepsy
NPY
Intracranial
I
1 Open
During M
Canavan disease
Aspartocyclase
Intracranial
I
1 Open/ 2 Completed
Seashore MR, Freeze A,
Leone P
Late infantile neuronal
ceroid lipofuscinosis
Tripeptydil peptidase
Intracranial
I
1 Open
Crystal R
Amyotrophic Lateral
Sclerosis
EAAT2
Intracranial
I
1 Under review
During M
Rheumatoid arthritis
TNFR:Fc
Intra-articular
I
2 Open
Mease P
hpAP
Intranasal/
Intrabronchial
I
2 Open
Aitken M
Neurological
diseases
Others
Gene marking
minidistrofine (~6-7 kb) e microdistrofine (~4 kb)
Queste versioni ridotte della distrofina presentano delezioni comuni della regione centrale a bastoncello e nel dominio C-terminale della
proteina parentale, lasciando intatti i domini funzionali essenziali della proteina, in particolare quello ricco in cisteine (CR)
Genetically engineered metabolic factory to treat genetic
diseases
Gene transfer by
AAV, Ad, LV, DNA vectors
Lung
Liver
Ectopic production of genetically engineered protein products (Factor IX,
Lysosomal enzymes, AAT-1, Epo, ecc)
Delivery into the circulation
Hemophilia
• Affects 1:5,000 males
– 80% hemophilia A due to Factor VIII deficiency
– 20% hemophilia B due to Factor IX deficiency
• Results in spontaneous bleeding, which can be fatal
• Treated with prophylactic or therapeutic infusion of the deficient factor
• Correction to 1% of normal activity would reduce spontaneous bleeding
• Correction to 10% of normal activity would eliminate most spontaneous bleeding
Muscle
Historic Overview on Hemophilia Therapy
Gene Therapy Approaches for Hemophilia:
Replacement therapy: blood transfusion since ‘70s
Recombinant FVIII/FIX infusion since ‘90s
• Liver-directed
• Muscle-directed
Gene Therapy Approaches for Hemophilia:
•
•
•
•
•
Existence of small (KO mice) and large (dog) animal models
Easy assessment of efficacy (coagulation tests)
Small correction should be sufficient (1%)
Liver-directed, Muscle-directed
Expensive therapy available
• Bone marrow-directed
• Transplantation of genetically-modified fibroblasts
Transplantation of genetically-modified
Fibroblasts
1, 2, 5 - FVIII (>8 kb)
3, 4 - FIX (1.4 kb)
working only after
hepatectomy
(in animals so far)
Gutless vectors?
One patient so far.
At 7 days: persistent elevation of transaminases = low therapeutic index
No additional patients enrolled
Blood, 2004
Intra-portal injection
Tail vein injection
M. Kay, K. High
Phase I Trial: 8 adult men with severe hemophilia B (<1%
baseline F.IX)
F.IX expression close to the site of injection up to 10 months after
treatment; no evidence for inflammation
Circulating levels of F.IX were less than what is required for
therapeutic effect
Lethal autosomal recessive disease
70 000 individuals affected worldwide;
1 in 3000 Caucasian births
High morbidity and mortality
F.IX activity assay and transaminase levels (AST, ALT) plotted as
a function of time in weeks after vector administration in subjects
E, F and G
CF Lung
The cystic fibrosis transmembrane conductance regulator (CFTR)
Normal Lung
Airway gene transfer
Adenovirus
In the absence of epithelial damage inefficient gene
transfer in CF patients
Cellular and humoral immune responses against the virus
Adeno-associated virus
Several trials carried out in the nose, sinus and single
lobes of CF patients (Vector from Targeted Genetics
Corp). Evidence of safety and limited efficacy
Repeated administrations feasible
Gene cloned in 1989
Natural variant serotypes more effective
First GT clinical trial in
1993
Nonviral gene transfer
Lipid-plasmid complexes w/wo chemical modifications
Offers several advantages over viral methods but are still
less efficient
Delivery of gene transfer vectors to lung: physical barriers and
innate immune responses
CF: gene therapy experience
>30 Clinical trial protocols, most of which completed
Strategies to enhance gene transfer to airway epithelia
1
Vector delivered from the basolateral surface
2
Transient tight junctions disruption
3
Modification of the apical membrane
4
New formulation and delivery methods
5
Engineered or natural vectors with improved
binding and entry properties
1
Mucus
2
Glycocalyx
3
Cilia
4
Macrophages
5
Humoral immune responses
Using the lung as a metabolic factory for secretion of therapeutic proteins
into the circulation
Factors limiting the duration of transgene expression in airway epithelia
1
Promoter shut-off
2
Loss of transgene from cells: particular problem for nonviral vectors
3
Bacterial DNA contained in plasmid vectors can generate
an inflammatory response
4
Immune response against viral vector or transgene
5
Limited life-span of differentiated airway cells
Vector administration to the lung might target the conducting airway epithelium, the alveolar
epithelium, or both. Secretion of protein products from the basolateral membrane of epithelial cells
must diffuse through the interstitium (composed of fibroblasts and connective tissue) and across
vascular endothelial cells, to be delivered into the blood (as marked by arrows). The alveolar
epithelia of the lung, which contain a prominent population of Type I pneumocytes, are in close
proximity to the lung capillary network, making them ideal gene therapy targets for systemic delivery
of proteins.