gene therapy of monogenic diseases - jagiellonian...

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Gene therapy of monogenic diseases

13 January 2014

Genetic diseases - incidence, mutated genes and detection rate

Disorder Incidence Gene Mutation detection rate

Monogenic Cystic fibrosis 1:4000 CFTR 98% Duchenne muscular dystrophy 1:3500 DMD ~90% Fragile X syndrome 1:4000 FMR 100% Huntington disease 1:5000-10 000 HD 100% Hemophilia A 1: 10 000 F8C ~90% Phenyloketonuria 1: 10 000 PAH 99% Polycystic kidney disease 1:1500 PKD1, PKD2 ~15%

Inherited cancer Breast-ovarian cancer 1:4000 BCRA1 (80%) 50-65%

BCRA2 (20%) 35% Li-Fraumeni syndrome p53 50% Ataxia-telangiectasia ATM 70% Familial polyposis coli 1:4000 APC 87% Hereditary non-polyposis coli 1:2000 MLH1 (30%) 33%

MLH2 (60%) 12% Cardiovascular disorders Familial hypercholesterolemia 1:500 LDLR 60% Hyperlipidemia APOE 10%

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Severe combined immunodeficiency

diseases

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First controlled trial of gene therapy - 1990

HPRT ADA

ADA deficiency– results in severe immunodeficiency syndrome

Gene therapy of ADA deficiency

ADA

ADA- ADA+

ADA+

Ashanti De Silva (patient)

First clinical trial of gene therapy - 1990

Retroviral vector containing correct ADA gene (cDNA) has been transduced into blood lymphocytes

This first clinical trial was not „pure” from the methodological point of view. The patients have been treated concomitantly with enzyme injections – ADA-PEG. Nevertheless, the marker transgene (neo) could be detected in the blood cells of the patients even more than 5 years after injection of modified cells.

Adult stem cells

hematopoietic stem cells

mesenchymal stem cells

bone marrow

HSC – hematopoietic stem cells

K.Szade, 2012

Szade et al., PLoS One, 17 May 2013

CD34+ positive cells are isolated from the patients’ bone marrow

Transduction of bone marrow stromal cells

Retroviral-GFP Adenoviral-GFP

Plasmid-GFP

Efficacy 40-60% Efficacy ~ 60%

AAV serotyp 6 (48h after tramsduction

Efficacy ~ 20%

Efficacy~ 5-10 %

Among 10 patients with ADA deficiency, restoration of immune functions has been achieved in 9

No serious side effects (leukemias) have been observed


X-linked severe combined immunodeficiency X-SCID

Lack of correct γc cytokine receptor gene

David Vetter

Cavazzana-Calvo M et al .

Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease

Science 2000: 28 April: 288: 669-672

X-linked severe combined immunodeficiency (X- SCID)

Restoration of B and T lymphocytes and NK cells

D. Kohn et al., Nature Rev Cancer July 2003

Lack of γc gene

SCID-X1: 1.  French trial – 10 treated, 9 benefited. Unfortunately, four of those

who benefited in the begining developed leukemia and one boy died this year because of leukemia.

2. British trial – 10 treated, 10 benefited –one developed leukemia

Gene therapy has been beneficial to most treated SCID-X1 and ADA

patients!!!

Potential risk of application of retroviral vectors

• long-term expression & integration into cellular genome

gag pol env ITR ITR

retrovirus

transgen ITR ITR

Retroviral vector

•  gag – structural proteins

•  pol – reverse transcriptase

•  env – envelope proteins

random integration – risk of insertional mutagenesis

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Integration of retroviral vector into the promoter of LMO2 gene

Serious side effects of SCID-X1 gene therapy

- development of uncontrolled clonal T lymphoproliferative syndrome, similar to acute lymphoblastic leukemia (ALL) in 4 out of 10 treated children in Paris and 1 boy treated in London

-  due to the integration of a vector into an LMO2 gene either close to the promoter or in the first intron Reasons: 1. LMO-2 locus is one of sites for retroviral integration

2. Cells with aberrant expression of LMO-2 could have been selected because the provide a clonal growth advantage

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Side effects of MMLV-based retroviral vectors prompted investigations of the mechanisms of integration and search for the new, safer vectors

Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency characterized by recurrent, often life threatening bacterial and fungal infections due to a functional defect in the microbial-killing activity of phagocytic neutrophils. It occurs as a result of mutations in genes encoding a multicomponent enzyme complex, the NADPH oxidase, that catalyses the respiratory burst. The majority of patients have an X-linked form of the disease which is associated with mutations in a membrane-bound component gp91phox. HLA-matched allogeneic hematopoietic stem cell (HSC) transplantation can be curative, but for patients without suitable donors, genetic modification of autologous hematopoietic stem cells is an attractive alternative.

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Chronic granulomatous disease

Dr Manuel Grez’s website 22 Gp91phox

Nature Medicine April 2006 23

Correction of neutrophil bacteriocidal function by overexpression of gp91phox

subunit of NADPH oxidase

Naldini L, Nature Med. April 2006 24

Side effects due to insertional mutagenesis occured also In gene therapy for X-linked chronic granulomatous disease

Quasim et al., Gene Therapy 2009 25

Blood. 2010 Jan 28;115(4):783-91. Epub 2009 Dec 1. Retrovirus gene therapy for X-linked chronic granulomatous disease can achieve stable long-term correction of oxidase

activity in peripheral blood neutrophils. Kang EM, Choi U, Theobald N, Linton G, Long Priel DA, Kuhns D, Malech HL. Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA. [email protected] Chronic granulomatous disease (CGD) is associated with significant morbidity and mortality from infection. The first CGD gene therapy trial resulted in only short-term marking of 0.01% to 0.1% of neutrophils. A recent study, using busulfan conditioning and an SFFV retrovirus vector, achieved more than 20% marking in 2 patients with X-linked CGD. However, oxidase correction per marked neutrophil was less than normal and not sustained. Despite this, patients clearly benefited in that severe infections resolved. As such, we initiated a gene therapy trial for X-CGD to treat severe infections unresponsive to conventional therapy. We treated 3 adult patients using busulfan conditioning and an MFGS retroviral vector encoding gp91(phox), achieving early marking of 26%, 5%, and 4% of neutrophils, respectively, with sustained long-term marking of 1.1% and 0.03% of neutrophils in 2 of the patients. Gene-marked neutrophils have sustained full correction of oxidase activity for 34 and 11 months, respectively, with full or partial resolution of infection in those 2 patients. Gene marking is polyclonal with no clonal dominance. We conclude that busulfan conditioning together with an MFGS vector is capable of achieving long-term correction of neutrophil oxidase function sufficient to provide benefit in management of severe infection.

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SCID-X1 (most common) 1.  France – 10 treated, 9 efficient 2.  Great Britain – 10 treated, 10 benefited

ADA deficiency (without ADA-PEG supplementation) 1.  Italy - 15 treated , 13 benefited 2.  Great Britain – 5 treated, majority benefited

children with ADA deficiency, treated with gene therapy, did not develop leukemias

Gene therapy appears to be effective in treatment of children with immunodeficiency

Other immunodeficiencies treated with gene therapy:

chronic granulomatous disease Wiskott-Aldrich disease

Gene therapy of Wiskott-Aldrich syndrome

P. Leboulch, Nature, 15 August 2013


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I. Verma, Science 23 August 2013

Gene therapy of metachromatic leukodystrophy

Side effects in clinical trials of gene therapy

X-SCID: 5 cases of acute lymphoblastic leukemia – (25% treated boys) – one patient died

X-linked chronic granuomatous disease:

4 cases of myelodysplastic syndrome (a pre-leukemic condition) and monosomy 7 in 3 of those 4 patients (two children, two adults – adults have died

Wiskott-Aldrich syndrome – 2 cases of leukemia

There is no therapy without side effects!

Quasim et al., Gene Therapy 2009

Gene therapy of immunodeficiency diseases

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Adrenoleukodystrophy (ALD)

People with ALD accumulate high levels of saturated, very long chain fatty acids in their brain and adrenal cortex because the fatty acids are not broken down by an enzyme in the normal manner. The biochemical pathogenesis that leads to the massive demyelination The gene responsible for X-linked ALD has been cloned and shown to be an ABC transporter protein. To date, the substrate transported by the ALD protein and the relationship between its transport function and VLCFA-CoA synthase activation are unknown

This X-linked recessive disease, with an estimated frequency of 1/20,000 men, presents in a variety of phenotypes [24]. In the most severe late infantile or juvenile cerebral form, which has a mean age of onset of about 7 years and constitutes 40 to 50% of the cases, neurological symptoms predominate. Initial behavioral and school problems are followed by gait disturbances, visual and hearing impairment, varying alterations of cognitive functions with progressive dementia and a devastating downhill course toward an apparent vegetative state in 3 to 5 years.

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Treatment of adrenoleukodystrophy

1.  Bone marrow transplantation 2.  Lorenzo’s oil

1984 r. Michaela & Augusto Odone (in movie played by Susan Sarandon & Nick Nolte)

4:1 mixture of glyceryl trioleate and glyceryl trierucate (so called: Lorenz’s oil) in combination with a diet low in VLCSFA (very long chain saturated fatty acids), have been used with limited success, especially before disease symptoms appear

X-linked adrenoleukodystrophy – accumulation of large amounta of very long chain saturated fatty acids – leads to demyelination and early death

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Gene therapy of adrenoleukodystrophy

40 Science, 6th November 2009

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Gene therapy of adrenoleukodystrophy

Brain MRI

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Thalassaemia 1.Autosomal inherited blood disorder 2. Patients have defects in either alpha or beta globin chain gene (unlike sickle-cell

disease) – abnormal red blood cells are produced 3. Therapy for thalassaemia primarily involves chelation or removal of excessive

iron from the blood

- patients with severe thalassaemia require blood transfusion

- bone marrow transplant (BMT) from compatible donor (sibling’s) - BMT from haploidentical mother to child

Treatment of beta-thalassemia

Persons, Nature September 2010 -self-inactivating lentiviral vector 43

Nature, September 2010 44

Succesful application of gene transfer with retroviral/lentiviral vectors

Adenosine deaminase deficiency X-linked severe combined immunodeficiency Chronic granulomatous disease Wiskott-Aldrich syndrome

Adrenoleukodystrophy Metachromatic leukodystrophy

β-thalassaemia

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AAV vectors in clinical gene therapy trials

Leber’s congenital amaurosis – gene therapy 1.  Most common cause of congenital blindness in

children 2. LCA2 – one of the forms – caused by mutation in the

retinal pigment epithelium-specific 65-kD protein gene (RPE65)

3. RPE65 is required to keep light-sensing

photoreceptor cells – the rodes and cones of the retina – in operating order

4. The RPE65 gene encodes for the isomerohydrolase

that isomerizes bleached all-trans-retinal into photosensitive 11-cis-retinal (Jin et al., 2005; Moiseyev et al., 2005). If no 11-cis-retinal is produced due to loss of or impaired RPE65 function, the chromophore rhodopsin cannot be assembled, and the photoreceptors remain insensitive to light stimuli

5. LCA2 is a rare diseases – in USA only 2000 people –

but is untreatable and causes blindness early in life 47

Lancet, October 2009 48

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First registered gene therapy drug in Europe

Glybera – first registered AAV vector for human gene therapy

Lipoprotein lipase deficiency – first officialy registered drug

– Glybera – accepted by European Commission – 25.10.2012

AAV vector with cDNA of lipoprotein lipase (LPL)

A centralised EU marketing authorisation has been obtained under the name Glybera on with the number EU/1/12/791

Gene therapy is effective in a number of monogenic diseases 1. Immunodeficiencies

- X-SCID immunodeficiency: retroviral vectors & hematopoietic stem cells - ADA- immunodeficiency - retroviral vectors & hematopoietic stem cells - chronic granulomatous diseases - retroviral vectors & hematopoietic stem cells

2. Congential blindness:

- Leber’s congenital amaurosis – rAAV vectors 3. Metabolic diseases - lipoprotein lipase deficiency - first registered drug Some beneficial effects have been observed in treatment of:

1. Adrenoleukodystrophy – lentiviral vector & hematopoietic stem cells 2. β-thalassemia – lentiviral vector & hematopoietic stem cells

SUMMARY

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Successful gene therapies

Science, 7th October 2011

Disease targets for gene therapy

Disease Gene(s)

Cystic fibrosis CFTR, α-1-anti-trypsin Gaucher disease glucocerebrosidase Hemophilia A Factor VIII Hemofilia B Factor IX Familial hypercholesterolemia LDL-R Muscular dystrophy sarcoglycan, dystrophin,

utrophin Ornithine transcarbamylase deficiency OTC

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Hemofilia A and B and gene therapy

1. Factor VIII production is not regulated in response to bleeding 2. The broad therapeutic index of factor VIII minimises the risk of

overdoses 3. Delivery of factor VIII into the bloodstream does not require

expression of the gene by specific organ 4. Even low levels of the protein can be beneficial

mRNA - factor VIII - 8,8 kb factor IX - 1,8 kb

Size of the coding sequences of factor VIII and factor IX

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Steps in human factor VIII gene transfer procedure

Roth DA et al., NEJM 2001; 344: 1735

Clinical gene therapy for haemophilia A

Ex vivo – plasmid gene therapy

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Roth DA et al., NEJM 2001; 344: 1735

Clinical gene therapy for haemophilia A

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Bleeding Events and Use of Exogenous Factor VIII in Three of the Six Patients.

Roth DA et al., NEJM 2001; 344: 1735 57

Disease targets for gene therapy Disease Gene(s)

Cystic fibrosis CFTR, α-1-anti-trypsin Gaucher disease glucocerebrosidase Hemophilia A Factor VIII Hemofilia B Factor IX Familial hypercholesterolemia LDL-R Muscular dystrophy sarcoglycan, dystrophin,

utrophin Ornithine transcarbamylase deficiency OTC

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FIX deficiency – so called Christmas disease

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1.  Self-complementary AAV vector 2.  AAV8-pseudotyped 3.  Targeting to liver 4.  Limitations of immune response (lower prevalence of anti-AAV8 antibodies)

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Is gene therapy for hemophilia necessary?

gene therapy of monogenic diseases - jagiellonian...

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