Géntherápia:
Még mindig csak álom vagy realitás?
Molnár Mária Judit
Molekuláris Neurológiai Klinikai és Kutatási Központ
Semmelweis Egyetem
Magyar Személyre Szabott Medicina Társaság 2. Konferenciája 2011. 09. 23.-24.
The history of the gene therapy
1970 1977 1990 1999 2000 2006 2006 2009 -
S. Rogers: gene therapy to treat 2 sisters whith argininemia
A gene was successfully delivered into mammalian cells
First human gene therapy was approved: SCID
J. Gelsinger with OTC deficiency died from organ failure
after gene therapy
A. Fischer cured children with SCID using retroviral vector,
2 of the children developed leukemia. FDA halted the use
of retroviruses in the US
Patients was successfully treated with metastatic
melanoma using killer T cells genetically retargeted to
attack the cancer cells
Succesfull gene-based th. for the treatment of HIV:
lentiviral vector for delivery of an antisense gene againts
HIV envelope
Researchers succeeded at halting adrenoleukodystrophy,
using a vector derived from HIV to deliver the gene for the
missing enzyme
The number of gene therapeutical clinical trials
Phase I
N:1023
Phase I/I
N:342
Phase II
N:1237
Phase II/III N:214
Phase III
2006 N:1064
N:562
2011 N:3398
General applications of therapeutic gene transfer
1. Molecular therapy for genetic diseases
2. Establishment of a stable gene reservoir as a
source of therapeutic proteins in non - genetic
diseases
3. To increase the tumor cells sensitivity to drugs
4. Destruction of malignant cells in neoplasias
5. DNA vaccines
Therapeutic options in genetic disorders
MUTANT GENE1>> MUTANT RNA2 > > DEFECTIVE PROTEIN3 PHENOTYPE4
Therapeutic targeting of either 1+2+3+4: Classical Pharmacology
Therapeutic targeting of 1 : DNA editing, Gene transfer, Gene replacement
Therapeutic targeting of 2 :Exon skipping, translational read-through, siRNA
Therapeutic targeting of 3 : Protein replacement, Protein expression
Therapeutic targeting of 4 : Pharmacology, physiotherapy, surgery
Targeting miscalleneous items : Enhancement of regeneration, tissue replacement
by cell therapy
Gene delivery
Vectors
Strategies
Viral
Retro
Herpes
Adeno
AAV
Lenti
Nonviral
Plasmid
Arteficial Chr
Liposomes
DNAsomes
Nanoparticlees
Molecular therapies
DNA modulating therapies

Gene replacement – to replace the defected gene

Gene transfer – to upregulate therapeutic protein

Gene editing – to correct the defected gene

Gene shifting – to upregulate healthy mtDNA molecules, change ratio of
heteroplasmy
RNA modulating therapies

Exon skipping by antisense oligonucleotides

Exon inclusion by antisense oligonucletotides

Mutant RNA removal

Inactivation of the mutant mRNA by RNAi

Destruction of the mutant mRNA by ribozymes

Translational manipulation
Protein modulating therapies

Neutralization of the mutant protein

Regulating the level of the haploinsufficient protein

Upregulating compansatory molecule
Clinical trials with DNA modulation
(using viral vectors)

Adrenoleukodystrophy

Alpha Sarcoglycan Deficiency

Gamma Sarcoglycan Deficiency

Late Infantile Neuronal Ceroid Lipofuscinosis:
AAVrh 10.- CLN2 gene Phase I/II

Pompe Disease: rAAV1-CMV-GAA phase I/ II
Adrenoleukodystrophy - 2009
Ex vivo viral correction







Bone marrow stem cells - CD34+ cells isolated
Ex vivo reactivation with cytokine míxture
Transfection by lentivirus containing a normal copy
of the ABCD1 gene
Patients were treated with CYC and busulfan
so that their hemopoetic stem cells containing the
ABCD1 mutation were greatly diminished.
Patients were then infused with their own CD34+ cells
engineered to contain normal copies of the ABCD1 gene
The engineered CD34+ can differentiate into a vast
number of different blood cell types.
They turned into microglial cells and corrected the
deficiencies in the brain responsible for X-ALD
Aubourg and Cartier-Lacave 2009
Gamma-SarcoGlycan Restoration
in LGMD2C - Phase I
Benveniste et al. AAN 2011. 04. 13.






Mid-to-high dosages of gamma-sarcoglycan
(SGC) genes in AAV1
9 participants, im injection extensor carpi
radialis muscle
– The first 2 groups received a single
injection
– The 2. group received a higher dose than
the first group
– The 3. group received 3 injections for the
highest total dose.
The therapy was well tolerated
In biopsy specimens 30 days after the inj.
showed SGC expression in 5/9 patients, with 4.7
to 10.5% of positively stained fibers and
detection of SGC mRNA by RT-PCR in the 3
patients who received the highest dose.
The SGC protein became also detectable by
Western blot in one patient
All patients seropositive for AAV1, 1 developed
a cytotoxic response again AAV1 capsid
In Duchenne MD
mdx mouse experiments
BUT!
• It is highly immunogenic
• Does not integrate in the
host genome
• Poor uptake into mature
muscle fibers - due to the
paucity of primary (CAR)
and secondary receptors
(integrin dimer) at the
mature cell surface
Numerous dystrophin positive fibers
after injection of AV-Dys
Deol et al. Mol Ther. 2007; 10:1767-74
Plasmid Mediated
Gene Transfer (PMGT)
Production
- easy
- cost effective
Non toxic
Week immunogeneicity
Plasmid - circular DNA that can carry
therapeutic genes into muscle
Inefficient uptake to
muscle fibers, unless
- electroporated or
- sonoporated
Electroporation (ELP) and Sonoporation (SP) Assisted
Plasmid Mediated Gene Transfer (PMGT)
 Factors influencing the transfection efficacy of the
PMGT, as
– parameters of ELP, SP
C57BL/6
– plasmid size
– age of animals
– use of Hyaluronidase
CD1
– use of microbubbles
SCID
 The stability of plasmid DNA in muscle and the
longevity of transfection after ELP assisted PMGT
Molnar et al. Mol Ther. 2004; 10:447-55
Sonoporation – Phase I trial
Subjects: 5 healthy male volunteers
(Ethically approved by local regulatory agencies)
Injection:
0.75-1.5 ml
saline susp.
VIALMIX®
activation: 45 sec
Injection:
0.75ml Definity
in saline susp.
Sonoporation:
Surface electrode
Frequency: 1 MHz
Duration: 2 min
Duty cycle: 30%
Intensity: 2-3 W/cm2
Sonoporation:
Surface electrode
Frequency: 1 MHz
Duration: 2 min
Duty cycle: 30%
Intensity: 2-3 W/cm2
SonoEnGene ®
Customed designed
ultrasound generator
Molnar et al. Neurology 2009; 72: 306-307
36 hours after injection and sonoporation
Molecular therapies
DNA modulating therapies

Gene replacement – to replace the defected gene

Gene transfer – to upregulate therapeutic protein

Gene editing – to correct the defected gene

Gene shifting – to upregulate healthy DNA molecules, change ratio of
heteroplasmy
RNA modulating therapies

Exon skipping by antisense oligonucleotides

Exon inclusion by antisense oligonucletotides
Mutant RNA removal
Inactivation of the mutant mRNA by RNAi
Destruction of the mutant mRNA by ribozymes
Translational manipulation




Protein modulating therapies

Neutralization of the mutant protein

Regulating the level of the haploinsufficient protein

Upregulating compansatory molecule
EXON SKIPPING





A mutation-specific therapy
Providing personalized medicine
Simultaneously may correct all isoforms
Maintains the original tissue-specific gene regulation
The antisense compounds inducing exon skipping are
small synthetic, and highly sequence-specific
- 2-O-methyl phosphorothioate (2OMe)
- phosphorodiamidate morpholino oligomers PMO.
- peptide-linked PMO
Exon skipping in Duchenne MD
13% of DMD patients: correct deletions of 50, 52, 45-50, 48-50, 49-50exons
Progress in AON exon skipping therapy in DMD
Timelines
PRO051
Prosensa/GSK
2OME AON
2007 2008 2009 2010 2011 2012 2013
Ph I
Im.
Ph I/II
Systemic adm, iv, weekly
Ph I/II
Study extension.
Ph I
Non ambulant
Ph II
Dosing
Ph III
Efficacy
Eteplirsen
AVI
PMO AON
Ph I
Im.
Ph I/II
Syst. admin. sc, 3 w
Ph I/II
Dosing
Systemic administration of PRO051 in DMD
 Weekly sc inj.for 5 weeks in 12 patients, with 4 doses (0.5, 2, 4, 6 mg/kg)
 Patients entered a 12-week open-label extension phase, during which
they all received PRO051 at a dose of 6.0 mg/kg/ week
 RNA splicing and protein levels in the tibial anterior muscle were
assessed
 The most common adverse events
 irritation at the administration site
 mild proteinuria and increased urinary α(1)-microglobulin levels;
 PRO051 induced spec. exon-51 skipping at doses of 2 mg/kg or more
 New dystrophin expression was observed between cc. 60% - 100% of
muscle fibers in 10 of the 12 patients, which increased in a dosedependent manner
 After the 12-week extension phase, there was improvement of
35.2±28.7 m (from the baseline of 384±121 m) on the 6-minute walk
test
 48 week extension study: no improvement in 6MWD
Goemans et al N. Engl J Med 2011;364(16):1513-22.
Goemens et al. AAN, 2011. 04. 13
Exon skipping trial in DMD with
AVI-4658 (eteplirsen)
• Eteplirsen was delivered iv. to 19 DMD
boys with mutations in the dystrophin
gene that can potentially benefit from
skipping of exon 51
• 12 weekly infusion (0·5, 1, 2, 4, 10, 20 mg/kg)
• Participants tolerated all doses well
• Muscle biopsies were performed at
baseline and at 14 weeks
• Patients were followed for 26 weeks
Cirak et al Lancet 2011 Aug;378(9791):595-605
Results: Lancet 2011 Aug 13; 378(9791):595-605
P15
Pre P15
P17
P18
P17
P19
P18
Post
Post Pre
Post
Pre
Post Pre Post Pre
• 7 patients responded to treatment dose higher than 2 mg/kg
• The dystrophin-associated proteins α-sarcoglycan and nNOS restored
• Analysis of the inflammatory infiltrate indicated a reduction of
cytotoxic T cells in the post-treatment muscle biopsies
in the two high-dose cohorts
Molecular therapies
DNA modulating therapies

Gene replacement – to replace the defected gene

Gene transfer – to upregulate therapeutic protein

Gene editing – to correct the defected gene

Gene shifting – to upregulate healthy DNA molecules, change ratio of
heteroplasmy
RNA modulating therapies

Exon skipping by antisense oligonucleotides

Exon inclusion by antisense oligonucletotides

Mutant RNA removal

Inactivation of the mutant mRNA by RNAi

Destruction of the mutant mRNA by ribozymes

Translational manipulation
Protein modulating therapies

Neutralization of the mutant protein

Regulating the level of the haploinsufficient protein

Upregulating compansatory molecule
siRNA therapy - Phase I trial – ALN-TTR01 iv.
Transthyretrin mediated amyloidosis




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

ALN-TTR blocks pathogenic accumulations of
mutant TTR in peripheral tissues (95% reduction
of V30M hTTR deposition)
Delivery next generation liponanoparticle (LNP)
2010-2012
Indication: FAP, FAC
Safety study
26 patients or carriers
Monitoring serum TTR level
Molecular therapies
DNA modulated therapies

Gene replacement – to replace the defected gene

Gene transfer – to upregulate therapeutic protein

Gene editing – to correct the defected gene

Gene shifting – to upregulate healthy DNA molecules, change ratio of
heteroplasmy
RNA modulating therapies

Exon skipping by antisense oligonucleotides

Exon inclusion by antisense oligonucletotides

Mutant RNA removal

Inactivation of the mutant mRNA by RNAi

Destruction of the mutant mRNA by ribozymes

Translational manipulation
Protein modulating therapies

Neutralization of the mutant protein

Regulating the level of the haploinsufficient protein

Upregulating compansatory molecule
~13% of boys have DMD due to
a nonsense mutation
Nonsense
(Premature
Stop) Codon
Dystrophin mRNA
Normal
Stop
Codon
Incomplete
dystrophin
K Bushby _ICNMD_2010
Ataluren – ns mutation codon read through
Nonsense
Normal
(Premature Stop) Codon Stop Codon
Dystrophin mRNA
Ataluren induces full-length protein production
Nonsense
(Premature Stop) Codon
Incomplete
dystrophin
Normal
Stop Codon
YIELD
Dystrophin mRNA
Full-length
dystrophin
K Bushby _ICNMD_2010
PTC – Genzyme
ATALUREN – Phase IIb

2010 March

A PTC Therapeutics failed in the Phase IIb trial
Tested patients: DMD with nonsense dystrophin mutation
Primary endpoint: 6 Min Walking Test – did not increased
significantly during the 48 weeks treatment period


BUT!

In CF the Phase II trial was successfull, Phase III trial is ongoing

Nonsense mutation hemophilia A & B (nmHA/B) and nonsense
mutation methylmalonic acidemia (nmMMA) Phase II trials are
ongoing as well.
Molecular therapies
DNA modulating therapies

Gene replacement

Gene editing

Gene shifting
RNA modulating therapies

Exon skipping by antisense oligonucleotides

Mutant RNA removal

Block RNA expression

Inactivation of the mutant mRNA by RNAi

Destruction of the mutant mRNA by ribozymes

Translational manipulation
Protein modulating therapies

Neutralization of the mutant protein

Regulating the level of the haploinsufficient protein

Upregulating compansatory molecule
Utrophin upregulation



Daily treatment with
SMTC1100, a novel small
molecule utrophin
upregulator, dramatically
reduces the dystrophic
symptoms in the mdx mouse
- K Davies group
Phase I trial – present
formulation results in low
plasma level
Formulation must be
improved
Tinsley et al: PLoS One. 2011; 6(5): e19189.
General applications of therapeutic gene
transfer
1. Molecular therapy for genetic diseases
2. Establishment of a stable gene reservoir as a
source of therapeutic proteins in non - genetic
diseases
3. To increase the tumor cells sensitivity to drugs
4. Destruction of malignant cells in neoplasias
5. DNA vaccines
Neurodegenerative Disorders

Alzheimer Disease A Phase II Study to Assess efficacy of
CERE-110 [Adeno-Associated Virus based, VectorMediated Delivery of Beta-Nerve Growth Factor (NGF)] in–
Ceregene

Parkinson disease
- Restoration the dopamin level of the basal ganglia
- To prevent or modify the secondary changes
due to the dopamin deficiency (Kaplitt procedure)
The Kaplitt Experiment
Tp “calm down” the overactive neurons of the subthalamic nucleus which
gives rise to signals that cause bradykinesia and tremor in Parkinson’s
disease
AAV2-GAD gene therapy for
advanced Parkinson's disease:
a double-blind, randomised trial

55 patients were randomised

Significant improvements in UPDRS score were noted in
the experimental group compared to sham in the offmedication motor scores
 At the 6-month endpoint, UPDRS score for the AAV2-GAD
group decreased by 8.1 points ( p<0·0001) and by 4.7 points
in the sham group (p=0·003).
 The AAV2-GAD group showed a significantly greater
improvement from baseline in UPDRS scores compared
with the sham group over the 6-month

Side effects: mild or moderate, likely related to surgery
and resolved. The most common were headache (7:2) and
nausea (6:2)
LeWitt Lancet Neurol. 2011 Apr;10(4):309-19
Phase I. Clinical Trial Gene Therapy for Pain

10 patients with intractable focal pain

Vector: replication-defective HSV-based vector expressing
human preproenkephalin (PENK)

Delivery: intradermal injection corresponding to the
distribution of the pain

Subjects receiving the low dose of NP2 reported no substantive
change in pain

Subjects in the midle- and high-dose cohorts reported pain
relief as assessed by numeric rating scale and SF-MPQ
Fink et al. Ann Neurol 2011 Aug;70(2):207-12.
General applications of therapeutic gene
transfer
1. Molecular therapy for genetic diseases
2. Establishment of a stable gene reservoir as a
source of therapeutic proteins in non - genetic
diseases
3. To increase the tumor cells sensitivity to drugs
4. Destruction of malignant cells in neoplasias
5. DNA vaccines
Malignant high grade glioma
Phase III Trial

Antisense oligonucleotide trabedersen

Designed for the specific inhibition of TGF-β2 synthesis
 TGF-β2 is overexpressed in more than 90% of highgrade gliomas, its levels are closely related to tumor
progression
 Inhibition of TGF-β2 in tumor tissue leads to
reversal of tumor-induced immune suppression,
inhibition of tumor growth and invasion
Curr Pharm Biotechnol 2011 May 27. [Epub ahead of print
Gene Therapy Is Still a Dream Or Reality?
2011
Gene therapy is not a failure —
it is simply too immature to deliver yet
on its promises
Theodore Friedman
Nature Med 1996; 2:144
THANK YOU FOR YOUR ATTENTION!