Géntherápia: Géntherápia: Még mindig csak álom vagy realitás?Még mindig csak álom vagy realitás?

Molnár Mária JuditMolnár Mária JuditMolekuláris Neurológiai Klinikai és Kutatási KözpontMolekuláris Neurológiai Klinikai és Kutatási Központ

Semmelweis EgyetemSemmelweis Egyetem

Magyar Személyre Szabott Medicina Társaság 2. Konferenciája 2011. 09. 23.-24.

The history of the gene therapyThe history of the gene therapy

1970 - S. Rogers: gene therapy to treat 2 sisters whith argininemia

1977 - A gene was successfully delivered into mammalian cells1990 - First human gene therapy was approved: SCID1999 - J. Gelsinger with OTC deficiency died from organ failure

after gene therapy2000 - A. Fischer cured children with SCID using retroviral

vector, 2 of the children developed leukemia. FDA halted the use of retroviruses in the US

2006 - Patients was successfully treated with metastatic melanoma using killer T cells genetically retargeted to attack the cancer cells

2006 - Succesfull gene-based th. for the treatment of HIV: lentiviral vector for delivery of an antisense gene

againts HIV envelope2009 - 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 The number of gene therapeutical clinical trials

2011 N:3398

2006 N:1064

Phase I N:1023

Phase I/I N:342

Phase II N:1237

Phase II/III N:214

Phase III N:562

General applications ofGeneral applications of therapeutictherapeutic gene transfergene 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 disordersTherapeutic 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

Vectors

Viral Nonviral

PlasmidArteficial Chr

LiposomesDNAsomes

Nanoparticlees

RetroHerpesAdenoAAVLenti

Strategies

Gene delivery

Molecular therapiesMolecular 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 modulationClinical trials with DNA modulation(using viral vectors)(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 Adrenoleukodystrophy - 2009

Ex vivo viral correctionEx 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 Gamma-SarcoGlycan Restoration in LGMD2C - Phase Iin LGMD2C - Phase I

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

Benveniste et al. AAN 2011. 04. 13.

Numerous dystrophin positive fibers after injection of AV-Dys

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

In Duchenne MD mdx mouse experiments

Deol et al. Mol Ther. 2007; 10:1767-74

Plasmid Plasmid Mediated Mediated Gene Transfer (PMGT) Gene Transfer (PMGT)

Production- easy- cost effective

Non toxicWeek immunogeneicity

Inefficient uptake tomuscle fibers, unless

- electroporated or- sonoporated

Plasmid - circular DNA that can carry therapeutic genes into muscle

Electroporation (ELP) and Sonoporation (SP) AssistedElectroporation (ELP) and Sonoporation (SP) AssistedPlasmid Mediated Gene Transfer (PMGT)Plasmid Mediated Gene Transfer (PMGT)

Factors influencing the transfection efficacy of the PMGT, as– parameters of ELP, SP– plasmid size – age of animals– use of Hyaluronidase– use of microbubbles

The stability of plasmid DNA in muscle and the longevity of transfection after ELP assisted PMGT

C57BL/6

CD1

SCID

Molnar et al. Mol Ther. 2004; 10:447-55

Sonoporation – Phase I trialSonoporation – Phase I trial

Injection:0.75-1.5 ml saline susp.

Injection:0.75ml Definityin saline susp.

Sonoporation:Surface electrodeFrequency: 1 MHzDuration: 2 minDuty cycle: 30%Intensity: 2-3 W/cm2

Molnar et al. Neurology 2009; 72: 306-307

Sonoporation:Surface electrodeFrequency: 1 MHzDuration: 2 minDuty cycle: 30%Intensity: 2-3 W/cm2

Subjects: 5 healthy male volunteers

(Ethically approved by local regulatory agencies)

VIALMIX®

activation: 45 sec

SonoEnGene ®

Customed designedultrasound generator

36 hours after injection and sonoporation36 hours after injection and sonoporation

Molecular therapiesMolecular 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

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 SKIPPINGEXON SKIPPING

Exon skipping in Duchenne MDExon skipping in Duchenne MD13% of DMD patients: correct deletions of 50, 52, 45-50, 48-50, 49-50exons13% of DMD patients: correct deletions of 50, 52, 45-50, 48-50, 49-50exons

Progress in AON exon skipping therapy in DMDProgress in AON exon skipping therapy in DMD

TimelinesTimelines 2007 2008 2009 2010 2011 2012 20132007 2008 2009 2010 2011 2012 2013

PRO051Prosensa/GSK

2OME AON

EteplirsenAVI

PMO AON

Ph IIm.

Ph I/IISystemic adm, iv, weekly

Ph I/IIStudy extension.

Ph INon ambulant

Ph IIDosing

Ph IIIEfficacy

Ph IIm.

Ph I/IISyst. admin. sc, 3 w

Ph I/IIDosing

Systemic administration of PRO051 in DMDSystemic 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 dose-dependent 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

• 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

Exon skipping Exon skipping trial in DMD with trial in DMD with AVI-4658 (eteplirsenAVI-4658 (eteplirsen))

Cirak et al Lancet 2011 Aug;378(9791):595-605

P15 P17 P18

P19

Pre

Post

Post Pre Post Pre Post Pre Post Pre

Results: Lancet 2011 Aug 13; 378(9791):595-605

P15 P17 P18

• 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 therapiesMolecular 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 - siRNA therapy - Phase I trial – ALN-TTR01 iv.Phase I trial – ALN-TTR01 iv.

Transthyretrin mediated amyloidosis

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 therapiesMolecular 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 ~13% of boys have DMD due to a nonsense a nonsense mutation mutation

Incompletedystrophin

Nonsense(Premature

Stop) Codon

NormalStop

Codon

Dystrophin mRNA

K Bushby _ICNMD_2010

AtalurenAtaluren – ns mutation codon read through – ns mutation codon read through

Incompletedystrophin

Nonsense(Premature Stop) Codon

NormalStop Codon

YIELD

Full-length dystrophin

NormalStop Codon

Nonsense(Premature Stop) Codon

Dystrophin mRNA

Ataluren induces full-length protein production

Dystrophin mRNA

K Bushby _ICNMD_2010

PTC – GenzymePTC – Genzyme ATALUREN – Phase IIb 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 therapiesMolecular 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 upregulationUtrophin 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 DisordersNeurodegenerative Disorders

Alzheimer Disease A Phase II Study to Assess efficacy of CERE-110 [Adeno-Associated Virus based, Vector-Mediated 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)

TheThe Kaplitt Kaplitt ExperimentExperiment

Tp Tp “calm down” the overactive neurons of the subthalamic nucleus which “calm down” the overactive neurons of the subthalamic nucleus which gives rise to signals that cause bradykinesia and tremor in Parkinson’s gives rise to signals that cause bradykinesia and tremor in Parkinson’s

diseasedisease

AAV2-GAD gene therapy for AAV2-GAD gene therapy for advanced Parkinson's disease: advanced Parkinson's disease: a double-blinda double-blind, randomised trial, randomised trial

55 patients were randomised

Significant improvements in UPDRS score were noted in the experimental group compared to sham in the off-medication 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 - Phase I. Clinical Trial - Gene Therapy for PainGene 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 ofGeneral applications of therapeutictherapeutic gene gene transfertransfer

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 gliomaMalignant high grade glioma

Antisense oligonucleotide trabedersen

Designed for the specific inhibition of TGF-β2 synthesis TGF-β2 is overexpressed in more than 90% of high-

grade 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

Phase III Trial

Gene Therapy Gene Therapy Is Still a Dream Or RealityIs Still a Dream Or Reality?? 20112011

Gene therapy is not a failure — Gene therapy is not a failure —

it is simplyit is simply tootoo immature to deliver yet immature to deliver yet

on its promiseson its promises

Theodore Friedman Nature Med 1996; 2:144

THANK YOU FOR YOUR ATTENTION!