Gene Therapy Monogenic Deficiency Diseases Regenerative Medicine Summerschool Niek van Til, July 4, 2017

What is gene therapy?

What is gene therapy?

the introduction of normal genes into cells in place of missing or defective ones in order to correct genetic disorders.

Rare diseases In humans, more than 8000 inherited diseases have been identified; in around 40% the genetic defect has been identified An estimated 30 million people in the European Union are affected by a rare disease Human suffering as well as health care costs are immense, and therefore the development of curative therapy is a humanitarian, medical and societal necessity In patient numbers, the inherited diseases are most frequent in disorders of the blood cell producing system

Rare, inherited diseases In humans, more than 8000 inherited diseases have been identified; in around 40% the genetic defect has been identified Approaches:

•prenatal diagnosis genetic counseling

•symptomatic therapy

•replacement therapy

•gene therapy A single mutation in the code may have profound

effects at the level of the organism

Rare, inherited diseases In humans, more than 8000 inherited diseases have been identified; in around 40% the genetic defect has been identified Current genetic therapy development:

•Hemophilia •Inherited immune deficiencies •Storage disorders (e.g. Hurler and Pompe diseases) •Duchenne’s muscular dystrophy •Sickle cell anemia •Thalassemia

A single mutation in the code may have profound

effects at the level of the organism

Which gene delivery system should we use?

Gene therapy vehicles

• Non-viral vectors

• Viral vectors

Gene therapy vehicles

• Adenoviral vectors

• Adeno-associated viral (AAV) vectors

• Retroviral vectors

Gene therapy vehicles

• Adenoviral vectors (in vivo GT)

• Adeno-associated viral (AAV) vectors (in vivo GT)

• Retroviral vectors (ex vivo GT)

‘The Royal disease’

Hemophilia A

AAV vectors

• Hemophilia (1:5,000 male newborns: generally common for an inherited disease)

• Hemophilia A and B (FVIII and FIX)

• Co-agulation defect.

• Recombinant protein infusions

• Develop antibodies/inhibitors/is expensive/not curative.

Recombinant AAV2 Manno et al

Recombinant AAV2/8

Nathwani et al

Lipoprotein lipase deficiency

• Enzyme that clears triglycerides from plasma

• AAV1-LPL enzyme

Glybera® (alipogene tiparvovec)

• First gene therapy product approved

• In October 2012, the European Commission granted marketing authorization for Glybera® under exceptional circumstances as a treatment for adult patients diagnosed with familial lipoprotein lipase deficiency (LPLD) confirmed by genetic testing, and suffering from severe or multiple pancreatitis attacks despite dietary fat restrictions.

Gene therapy vehicles

• Adenoviral vectors (in vivo GT)

• Adeno-associated viral (AAV) vectors (in vivo GT)

• Retroviral vectors (ex vivo GT)

Retroviridae (gammaretroviral vectors)

Targets: hematopoietic stem cell

Blood cell production

• Most blood cells live very short » White blood cells 12 hours » Platelets 8 days » Erythrocytes 3 months

• Every day an adult makes: 100,000,000,000 – 500,000,000,000 new blood cells, on average

100 gram = 1,100,000 – 5,700,000 per second

Blood cell production

• Most blood cells live very short » White blood cells 12 hours » Platelets 8 days » Erythrocytes 3 months

• Every day an adult makes: 100,000,000,000 – 500,000,000,000 new blood cells, on average

100 gram = 1,100,000 – 5,700,000 per second

• Stem cells located in bone marrow • These stem cells are easy to transplant: basis of clinical bone

marrow transplantation (mainly applied to treat leukemia)

White blood cells

Platelets

Red blood cells

blood

Bone marrow

thymus Re-infusion

Bone marrow puncture

Stem cells

Virus vector with gene

Cells with desired gene

THYMUS

CLP

NK

CD8

B

2.γc cytokine-dependent signals γc, JAK-3, IL7Ra

3.V(D)J recombination Rag-1/-2, Artemis

1.Prevention of cell apoptosis DNA replication (purin metabolism) ADA

HSC

Myeloid compartment

CD4

4.Pre TCR/TCR signalling) CD45, CD3δ,ε

SCID diseases

Slide by A. Fischer

SCID gene therapy

Bron: BBC News

SCID X1 as a paradigm for HSC gene therapy development

• Results superior to allogeneic stem cell transplantation both in efficacy as well as in over-all survival

• Complication: autonomous T cell clones leading to leukemia, elucidated as due to insertional mutagenesis resulting in leukemic transformation

Third generation lentiviral vector constructs - HIV derived:

Ψ ∆U3 R U5 Promoter Therapeutic Gene(s) p R U5 bPRE4* cPPT RRE

SIN LV vectors, Luigi Naldini Schambach A et al, Gene Ther. 2006

• Envelope protein: VSV-g

• Cellular promoters

Third generation lentiviral vector constructs - HIV derived:

Promoter Therapeutic Gene(s) LTR

SIN LV vectors, Luigi Naldini Schambach A et al, Gene Ther. 2006

• Envelope protein: VSV-g

• Cellular promoters

LTR

Lentiviral IL2RG Gene Therapy

Alternative to γ-retroviral vectors Lentiviral vectors have improved safety features due to their

distinct integration profile

– Able to integrate into quiescent cells…… – Deletion of enhancer regions from the LTRs reduces risk of

influencing neighbouring genes – Biased integration into actively transcribed genes but no bias

towards transcription start sites

Currently in preparation for clinical application

Gene therapy for lysosomal storage disorders

Glycogen storage disorder GSDII Pompe disease (Glycogenosis type II, acid maltase deficiency) Acid α-glucosidase deficiency

Gene therapy for lysosomal storage disorders

Clinical manifestations

Early onset Late onset

Progressive muscle weakness

Progressive muscle weakness

Enlarged heart / cardiomyopathy

Lordosis / scoliosis

Enlarged liver Sleep apnea

Respiratory function impairment

Respiratory function impairment

Failure to reach motor milestones

Frequent respiratory infections

No mental retardation!

Pompe disease: clinical spectrum

Classic infantile Late-onset

Center for Lysosomal and Metabolic Diseases Erasmus MC, Rotterdam, The Netherlands Van der Ploeg and Reuser, Lancet, 2008

Current attempts for therapy

Enzyme replacement therapy Not curative

Immune responses

Requires life-long administration

High costs

SF GAA bPRE4* cPPT

Infusion into recipient ♀ mouse

Hematopoietic Stem Cells

Virus with GAA gene

HSC with GAA gene

BM cells from ♂ mouse femurs

Gene Transfer Procedure: Gaa-/- mouse model

5’LTR 3’LTR

van Til et al, Blood, 2010

Infusion into recipient ♀ mouse

Hematopoietic Stem Cells

Virus with GAA gene

HSC with GAA gene

BM cells from ♂ mouse femurs

Gene Transfer Procedure: Gaa-/- mouse model

GAA bPRE4* cPPT 5’LTR 3’LTR

van Til et al, Blood, 2010

Partial correction of murine Pompe disease

van Til et al, Blood, 2010

Pompe stem cell gene therapy

30% of BM and blood cells express acid α-glucosidase, results: • High level of production of acid α-glucosidase • Restoration of acid α-glucosidase activity in tissues and

proportional reduction of glycogen storage • Full correction in liver and spleen • Full correction of the life-threatening cardiomyopathy • Significantly improved respiration • Improved, but not fully normalized skeletal muscle function Essentially at least as effective as enzyme replacement therapy, and: • Robust immune tolerance to the recombinant transgene product N.P.van Til*, M. Stok*, F.S.F. Aerts Kaya, T.P.Visser, E.Farahbakhshian, M.A. Kroos, M.C. de Waard, E.H. Jacobs, M.A. Willart, C.G. van der Wegen,

B.J. Scholte, B.N.M. Lambrecht, M.M.A. Verstegen, D.J. Duncker, A.T. van der Ploeg, A.J. Reuser, and G. Wagemaker. Lentiviral gene therapy of murine hematopoietic stem cells ameliorates the Pompe disease phenotype. Blood 2010 Jul 1;115(26):5329-37.

Improve protein production

Activity/integration

SF.GAA

SF.GAAco

SF.GFP

050

100150200250300350400450

(nm

ol/h

r/m

g)/in

tegr

atio

n

New vector with optimal sequence

Stok et al, unpublished data

02468

1012141618202224

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

days

dist

ance

(km

)

WTSF.GAAcoKO

Skeletal muscle correction – running wheels

Stok et al, unpublished data

02468

1012141618202224

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

days

dist

ance

(km

)

WTSF.GAAcoKO

Skeletal muscle correction – running wheels

Stok et al, unpublished data

Improve targeting to muscle

IGFII binds with high affinity to IGFII receptor

GAAco IGFIIco 3AA spacer

AA 70-952 SPS

ZC-701

1 and 8-67

Insulin-like growth factor II

IGFII receptor = CI-M6PR

Maga et al, JBC, 2013

Glycosylation-independent lysosomal targeting (GILT)

GILT-tagged GAA improves affinity to the IGF-II receptor 25-fold

Advantages: • HSC gene therapy currently the only approach to achieve both robust immune tolerance to the transgene product and effectively bypassing the blood/brain barrier

Future directions to improve safety and efficacy further: •Targeted gene delivery •Selective expression •Non-cytoreductive conditioning •Ex vivo stem cell expansion to promote engraftment of transduced cells

Stem Cell Gene Therapy

Acknowledgements Erasmus MC Hematology gene therapy: (Pompe)

Qiushi Liang Yvette van Helsdingen Merel Stok

Guus van der Velden Helen de Boer Marshall Huston Trudi Visser Fatima Aerts-Kaya Monique Verstegen

Gerard Wagemaker

Department of Clinical Genetics & Pediatrics Marian Kroos Arnold Reuser Ans van der Ploeg Pim Pijnappel

Department of Experimental Cardiology Monique de Waard Elza van Deel Dirk Duncker

Department of Cell Biology Pascal van der Wegen Bob Scholte

Department of Genetics Edwin Jacobs

Department of Lung diseases Bart Lambrecht

HSR-TIGET Luigi Naldini et al. MHH, Hanover Axel Schambach Christopher Baum

NCT and DKFZ Ali Nowrouzi Manfred Schmidt Christof von Kalle

Stefan Nierkens Jaap Jan Boelens Colin de Haar Maud Plantinga Niek van Til Lotte Spel Thijs Flinsenberg Nina Blokland Ester Dünnebach Marianne Boes Rick Admiraal Charlotte van Kesteren

U-DANCE Utrecht Dendritic Cells against Cancer

N.P.vanTil@umcutrecht.nl