Gene therapy of monogenic diseases

13 January 2014

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Genetic diseases - incidence, mutated genes and detection rate Disorder

Incidence

Gene

Mutation detection rate

1:4000 1:3500 1:4000 1:5000-10 000 1: 10 000 1: 10 000 1:1500

CFTR DMD FMR HD F8C PAH PKD1, PKD2

98% ~90% 100% 100% ~90% 99% ~15%

Breast-ovarian cancer

1:4000

Li-Fraumeni syndrome Ataxia-telangiectasia Familial polyposis coli Hereditary non-polyposis coli

1:4000 1:2000

BCRA1 (80%) BCRA2 (20%) p53 ATM APC MLH1 (30%)

50-65% 35% 50% 70% 87% 33%

MLH2 (60%)

12%

Monogenic Cystic fibrosis Duchenne muscular dystrophy Fragile X syndrome Huntington disease Hemophilia A Phenyloketonuria Polycystic kidney disease

Inherited cancer

Cardiovascular disorders 2 Familial hypercholesterolemia

1:500

LDLR

60%

Severe combined immunodeficiency diseases

3

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+

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.

Ashanti De Silva (patient)

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

bone marrow

mesenchymal stem cells hematopoietic stem cells

HSC – hematopoietic stem cells

K.Szade, 2012

CD34+ positive cells are isolated from the patients’ bone marrow Szade et al., PLoS One, 17 May 2013

Transduction of bone marrow stromal cells Plasmid-GFP

AAV serotyp 6 (48h after tramsduction

Efficacy~ 5-10 % Efficacy ~ 20% Adenoviral-GFP

Efficacy ~ 60%

Retroviral-GFP

Efficacy 40-60%

Gene therapy of ADA deficiency

Gene therapy of ADA deficiency

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) Lack of γc gene

Restoration of B and T lymphocytes and NK cells

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

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

Potential risk of application of retroviral vectors

• gag – structural proteins • pol – reverse transcriptase • env – envelope proteins ITR

gag pol env

• long-term expression &

integration into cellular genome ITR

retrovirus

random integration – risk of insertional mutagenesis ITR

transgen

ITR Retroviral vector

Integration of retroviral vector into the promoter of LMO2 gene

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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

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

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Chronic granulomatous disease 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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Dr Manuel Grez’s website

Gp91phox

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Nature Medicine April 2006

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Correction of neutrophil bacteriocidal function by overexpression of gp91phox subunit of NADPH oxidase

24 Naldini L, Nature Med. April 2006

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

Quasim et al., Gene Therapy 2009

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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 26 long-term correction of neutrophil oxidase function sufficient to provide benefit in management of severe infection.

Gene therapy appears to be effective in treatment of children with immunodeficiency 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 Other immunodeficiencies treated with gene therapy: chronic granulomatous disease Wiskott-Aldrich disease

Gene therapy of Wiskott-Aldrich syndrome

Gene therapy of Wiskott-Aldrich syndrome

P. Leboulch, Nature, 15 August 2013

Gene therapy of Wiskott-Aldrich syndrome

Gene therapy of Wiskott-Aldrich syndrome

I. Verma, Science 23 August 2013

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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!

Gene therapy of immunodeficiency diseases

Quasim et al., Gene Therapy 2009

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Adrenoleukodystrophy (ALD) 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. 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 37

Treatment of adrenoleukodystrophy X-linked adrenoleukodystrophy – accumulation of large amounta of very long chain saturated fatty acids – leads to demyelination and early death 1.  Bone marrow transplantation 2.  Lorenzo’s oil 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

1984 r. Michaela & Augusto Odone (in movie played by Susan Sarandon & Nick Nolte) 38

Gene therapy of adrenoleukodystrophy

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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

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Treatment of beta-thalassemia

-self-inactivating lentiviral vector

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Persons, Nature September 2010

44 Nature, September 2010

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

AAV vectors in clinical gene therapy trials

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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

48 Lancet, October 2009

First registered gene therapy drug in Europe

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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

SUMMARY 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

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Successful gene therapies

Science, 7th October 2011

Disease targets for gene therapy Disease

Cystic fibrosis Gaucher disease Hemophilia A Hemofilia B Familial hypercholesterolemia Muscular dystrophy Ornithine transcarbamylase deficiency

Gene(s)

CFTR, α-1-anti-trypsin glucocerebrosidase Factor VIII Factor IX LDL-R sarcoglycan, dystrophin, utrophin 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

Size of the coding sequences of factor VIII and factor IX mRNA - factor VIII - 8,8 kb factor IX - 1,8 kb 54

Clinical gene therapy for haemophilia A

Steps in human factor VIII gene transfer procedure

Ex vivo – plasmid gene therapy

Roth DA et al., NEJM 2001; 344:551735

Clinical gene therapy for haemophilia A

Roth DA et al., NEJM 2001; 344:561735

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

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

Disease targets for gene therapy Disease

Cystic fibrosis Gaucher disease Hemophilia A Hemofilia B Familial hypercholesterolemia Muscular dystrophy Ornithine transcarbamylase deficiency

Gene(s)

CFTR, α-1-anti-trypsin glucocerebrosidase Factor VIII Factor IX LDL-R sarcoglycan, dystrophin, utrophin OTC

FIX deficiency – so called Christmas disease 58

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1.  2.  3.  4. 

Self-complementary AAV vector AAV8-pseudotyped Targeting to liver 61 Limitations of immune response (lower prevalence of anti-AAV8 antibodies)

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Is gene therapy for hemophilia necessary?

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