Challenges to therapy for X-linked adrenoleukodystrophy

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Challenges to therapy for X-linked
adrenoleukodystrophy

• Nancy Braverman, MS, MD
• McGIll University-MCH-RI

March 11 2010 HGEN 171-575
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Peroxisomes originate from ER membranes and by
fission of existing peroxisomes
NEXT gtgt
Click to view animation gtgt
adapted from Annu Rev Genet. 200034623-652.
Sacksteder KA, Gould SJ.
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Role of peroxins in matrix protein import
Click to view animation gtgt
Gould, Raymond, Valle.In Metab Molec Basis of
Inh Dis. Ch 129 p. 3190.
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The 3 major metabolic pathways in peroxisomes
mitochondria
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Properties of peroxisomal matrix proteins

• Contain Peroxisome Targeting Sequences (PTS)
• Imported as oligomers/fully assembled proteins
• Can have dual localizations in mitochondria,
  cytosol

PTS1
PTS2
-SKL
R/KLX5Q/HL
-SKL
N - terminal (-R/KLX5 Q/HL-) Presequence
cleaved internally 3 enzymes only Thiolase,
PhyH, AGPS Receptor is PEX7
C - terminal (-SKL) Most matrix
proteins Receptor is PEX5
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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)

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Develop therapies targeted to the metabolic
defects

• Phytanic acid restriction
• Reduction in VLCFA
• dietary reduction
• enhance VLCFA omega oxidation
• reduce VLCFA synthesis
• Supplementation with DHA, bile acids,
  plasmalogens

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Develop therapies targeted to the molecular
defects

• Enhance activity of a defective PEX protein-
• improve 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?

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PBD phenotypes correlate with
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Mild PBD, PEX1-p.G843D/G843D
Px memb prot
CATALASE
PTS1/PTS2
37oC
30oC
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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
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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

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Intracellular distribution of AGT, a protein with
an N-terminal MTS C-terminal PTS1
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Targeting signal through evolution depends on diet
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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?
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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)

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Requirement for peroxisome functions in CNS
development
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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

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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

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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
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X-ALD defective peroxisomal ß-oxidation
Click to view animation gtgt
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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
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• adrenal insufficiency only
• Adult-onset cerebral involvement - dementia
• Female heterozygotes- 50 with milder AMN symptoms

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X-ALD pedigree ABCD1-c.1801delAG
Adrenal disease
0
Spastic paraparesis (AMN)
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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

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X-ALD treatment dietary changes

• Dietary therapy
• Restriction of dietary VLCFA intake
• Lorenzos oil- 41 mix
• Glycerol trioleate (C181)
• Glycerol trierucate (C221)
• Lowers plasma C260 and C240 levels
• Reduces, but does note eliminate the risk for the
  childhood leukodystrophy phenotype

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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)

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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

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X-ALD pedigree
Adrenal disease
5 months
0
Spastic paraparesis (AMN)
?
X-ALD
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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)

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Identifying modifier genes in X-ALD by SNP
association studies

• ABCD2 polymorphisms and clinical phenotypes
  showed an even allele distribution in different
  X-ALD phenotypes and controls
• Genes involved in methionine metabolism weak
  association with a polymporphism in Tc2

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ABCD1 null mouse model

• Has elevation of VLCFAs
• Does not develop cerebral disease
• Older mice develop axonal degeneration

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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 througho
ut the body
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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)

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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)

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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

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Hematopoetic stem cell lineages
Monocyte derived cells include brain microglial
cells
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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

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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 C260/C220 levels, grey band is normal
values
Long term expression in PBMCs, continue
expression in CD34 cells
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Analysis of LV insertion sites

• Showed multitude of insertion sites without
  clonal dominance

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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
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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

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