Download X linked adrenoleukodystrophy - Department of Medical Genetics

Challenges to therapy for Xlinked adrenoleukodystrophy
Nancy Braverman, MS, MD
McGIll University-MCH-RI
March 11 2010
HGEN 171-575
Peroxisomes originate from ER membranes
and by fission of existing peroxisomes
Click to view animation >>
adapted from Annu Rev Genet. 2000;34:623-652.
Sacksteder KA, Gould SJ.
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Role of peroxins in matrix protein import
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Gould, Raymond, Valle.In: Metab & Molec Basis of Inh Dis. Ch 129 p. 3190.
The 3 major metabolic pathways in
peroxisomes
mitochondria
Properties of peroxisomal matrix proteins
• Contain Peroxisome Targeting Sequences (PTS)
PTS1
PTS2
-SKL -SKL
C - terminal (-SKL)
Most matrix proteins
Receptor is PEX5
R/KLX5Q/HL
N - terminal (-R/KLX5 Q/HL-)
Presequence cleaved internally
3 enzymes only: Thiolase, PhyH, AGPS
Receptor is PEX7
• Imported as oligomers/fully assembled proteins
• Can have dual localizations in mitochondria, cytosol
Genetic disorders of peroxisomes
• Multiple enzyme deficiencies: Peroxisomal
Biogenesis Disorders (PBD)
– Zellweger spectrum disorder (ZSD) (~1/60,000)
– Rhizomelic chondrodysplasia punctata spectrum
(RCDP)(~1/100,000)
• Single enzyme deficiencies
– X-linked adrenoleukodystrophy (X-ALD) (~1/20,000)
– 3-methyl-CoA racemase deficiency
– Adult Refsum disease
– Hyperoxaluria Type I (Primary Hyperoxaluria)
Develop therapies targeted
to the metabolic defects
A--------->B
•
Phytanic acid restriction
•
Reduction in VLCFA
dietary reduction
enhance VLCFA omega oxidation
reduce VLCFA synthesis
•
Supplementation with DHA, bile acids, plasmalogens
Develop therapies targeted
to the molecular defects
• Enhance activity of a defective PEX proteinimprove protein folding
• Bypass the need for a specific PEX protein-
upregulate a partner PEX protein
• Induce peroxisome proliferation
• Enzyme/PEX protein replacement therapy ?
• Liver/bone marrow stem cell transplant ?
• Gene therapy ?
• Manipulate the intestinal microbiome?
PBD phenotypes correlate with:
 Severity of the biochemical defects
 Severity of import defect, peroxisome number &
size
 Predicted effect of the PEX gene mutation on
protein function
nonsense, frameshift, deletion alleles
no residual protein function
missense alleles some residual protein function
Mild PBD, PEX1-p.G843D/G843D
Px memb prot
37oC
30oC
PTS1/PTS2
CATALASE
Conformational changes of p97 AAA ATPase
during its ATPase cycle
Bind and hydrolyze ATP generating chemical energy that is converted into motion of
the molecule.
Motion used to pull PEX5 out of the membrane for another round of import
How do we improve protein folding?
• Lower temperature
• Chaperone (protein or drug)
– Nonspecific chemical chaperone
– Pharmacologic chaperone
Enzyme substrate
Protein/enzyme inhibitor
-protein kinase and kinase inhibitor
Vitamin cofactor
Intracellular distribution of AGT, a protein
with an N-terminal MTS & C-terminal PTS1
Targeting signal through evolution
depends on diet
Can we manipulate peroxisome
protein targeting for therapy?
Cell penetrating peptides:
Short sequence domains from known proteins that
are able to translocate across the plasma membrane
HIV-TAT, penetrin, octa-arginine
Fuse these domains to:
PTS1/PTS2 matrix protein
PEX proteins?
Better understanding of the disease
pathophysiology may reveal novel
targets for therapy: mouse models
 Selective inactivation of Pex5 gene in
neural cells (conditional gene targeting)
Requirement for peroxisome
functions in CNS development
X-linked adrenoleukodystrophy (X-ALD)
• Defect in adrenoleukodystrophy protein
(ALDP) encoded by the ABCD1 gene)
– Mapped to Xq28
– Over 200 mutations known, most result in no
detectable ALDP protein
• Incidence ~ 1/20,000
• All ethnic groups
• Reduced peroxisomal VLCFA oxidation
Clinical picture in a child with the
cerebral form
• Medical history
– 8-year old previously healthy, normal male
– Attention deficit/hyperactivity apparent within
the past year
– Performing poorly in 2nd grade
– Recently began to run clumsily and to walk stiffly
– No recent illnesses
– No medications
Rapid deterioration
in the X-ALD cerebral form
Deterioration in writing
over a 4 month period
Brain MRI –
white matter disease
Dec 29, 1989
Mar 5, 1990
May 3, 1990
X-ALD: defective peroxisomal
β-oxidation
Click to view animation >>
Multiple phenotypes of X-ALD
• Childhood cerebral form
~35%
– Onset - ~6-12 yrs (survival: several years)
– 90% with adrenal insufficiency
• Adrenomyeloneuropathy (AMN)
~50%
– Spastic paraparesis and sphincter dysfunction
– Onset - ~2nd-5th decade (survival: decades)
– 2/3 with adrenal insufficiency
• Other phenotypes
~15%
– adrenal insufficiency only
– Adult-onset cerebral involvement - dementia
• Female heterozygotes- 50% with milder AMN
symptoms
X-ALD pedigree: ABCD1-c.1801delAG
Adrenal disease
0
Spastic paraparesis (AMN)
Lack of genotype-phenotype
correlation in X-ALD
• All clinical phenotypes are observed in
the same nuclear family
• Deletion mutations are associated with
severe and mild phenotypes
• Monozygotic twins are reported with
different phenotypes
X-ALD treatment: dietary changes
• Dietary therapy
– Restriction of dietary VLCFA intake
– Lorenzo’s oil- 4:1 mix
• Glycerol trioleate (C18:1)
• Glycerol trierucate (C22:1)
– Lowers plasma C26:0 and C24:0 levels
– Reduces, but does note eliminate the risk for
the childhood leukodystrophy phenotype
X-ALD: other approaches to therapy
•
•
•
•
Cholesterol lowering drugs (Lovastatin)
Increase omega oxidation of VLCFA
Reduce endogenous elongation of VLCFA
Induce expression of partner proteins (the
anticonvulsant, valproate, induces ABCD2
expression)
X-ALD treatment: allogenic bone
marrow transplantation (from donor)
• Colonisation of the brain by cells of the
monocyte-macrophage system (become
microglial cells) provides the rationale for the
use of BMT in X-ALD
• Assymptomatic boys are put on LO and followed
closely for cerebral involvement
• At first signs of cerebral disease, a transplant is
recommended
X-ALD pedigree
Adrenal disease
0
X-ALD
Spastic
paraparesis
(AMN)
5 months
?
Environmental factors and
candidate modifier gene(s) in X-ALD
• Environmental factor as the initial trigger of
cerebral inflammation eg viral infection
• Genetic segregation analysis supports the role of
at least one autosomal dominant modifier gene
• Polymorphisms in ABCD2 (ALDPR), ABCD3
(PMP70), ABCD4 (PMP70R)
• Polymorphisms in genes encoding inflammatory
proteins
• Polymorphisms in elongation of VLCFA (ELOV1)
Identifying modifier genes in X-ALD
by SNP association studies
ABCD2 polymorphisms and clinical phenotypes
showed an even allele distribution in different XALD phenotypes and controls
Genes involved in methionine metabolism: weak
association with a polymporphism in Tc2
ABCD1 null mouse model
• Has elevation of VLCFAs
• Does not develop cerebral disease
• Older mice develop axonal degeneration
A comeback for gene therapy: ex
vivo gene correction
20 months
ALD patient
Reduction
in VLCFA
Gene-corrected HSCs
HIV-based vector with
therapeutic ABCD1 gene
Progeny of
gene-corrected
HSCs distribute
throughout
the body
Lentiviral vectors
• Retroviruses, adenoviruses, adeno-associated
viruses and lentiviruses are used in genetic
engineering.
• The most commonly used rLV vector is based on
the human immunodeficiency virus 1 (HIV-1)
Recombinant lentiviral gene therapy
vectors vs. other retroviral vectors
• Transduce HSCs more efficiently
• Self-inactivating long terminal repeats do
not promote transcription, thus reducing
the risk of mutagenesis and leukemia
• Infects non-dividing cells (neurons)
ABCD1 gene transfer with lentiviral
vectors: preclinical studies
• Cell model:
Transduced HsALDP- ,CD34+ cells (pluripotent
HSC)biochemical correction of derived
monocytes/macrophages
• Whole animal model:
Transduced MmALDP-, CD34+ cells into X-ALD
mice replacement of 25% brain microglial cells,
but mice do not devlop a leukodystrophy so cannot
tell if treatment is clinically effective
Hematopoetic stem cell lineages
Monocyte derived cells include brain microglial cells
2 patients (7 yo) with cerebral disease
with no matched donor
• CD34+ cells isolated, infected with HIV-1
lentiviral vector-ABCD1
• Patients underwent bone marrow ablation, in
order to repopulate the bone marrow with the
engineered cells
• Transduced cells were re-infused into the
patient without complications
Patient results: did it work?
A-C. Expression of ALDP in
PBMCs by IF using an ALDP
antibody
(CD14-moncytes, CD15
granulocytes, CD3 T
lymphocytes, CD19 B
lymphocytes)
D. Plasma C26:0/C22:0
levels, grey band is normal
values
Long term expression in PBMCs, continue expression in CD34+ cells
Analysis of LV insertion sites
• Showed multitude of insertion sites
without clonal dominance
Neurological outcomes
(A) Is patient 1 and (B) is
pateint 2before and after
gene therapy
(C) Is in an untreated
8-year-old ALD patient showing
the progression of cerebral
demyelinating lesions
Conclusions: is therapeutic
efficacy≅ allogenic BMT?
• Clinical outcome similar so far
• Data support HSC engraftment with capacity
to repopulate multiple hematopoetic
lineages
• Up to 14% of cells in each lineage expressed
ALDP in contrast to 100% in allogenic BMT
• However, ABCD1 gene was overexpressed by
its promoter, and this may have helped to
reduce VLCFA to equivalent levels