Download Current Therapy of Genetic Disorders

Current Therapy of Genetic
Disorders
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Preventive
Metabolic Manipulation
Gene Product Replacement
Cell or Organ Transplantation
Gene Therapy
Therapy of Genetic Disorders
• Preventive Therapy
– Prenatal diagnosis
– Preimplantation diagnosis
(in vitro fertilization, testing of embryo &
implantation of normal embryo)
– Preventive screening for disease onset
Therapy of Genetic Disorders
• Metabolic Manipulation
– Dietary restriction
• (Lactose restriction for Lactase deficiency;
phenylalanine restriction for phenylketonuria)
– Dietary Supplementation
• (Vitamin C for Scurvy, Biotin for Biotinidase
deficiency, Starch for G-6-P deficiency)
– Chelation and enhanced excretion
• (copper chelation for Wilson Disease)
– Metabolic inhibitors
• (allopurinol for gout, Statins for
hypercholesterolemia,)
Lactase deficiency:
Dietary Restriction: lactose free
(dairy products)
Dietary supplementation: lactase pills
All other mammals and most people lose the ability to digest
lactose by adulthood
Lactase persistence is found in 50-90% of Europeans but is
much rarer in other populations
Lactase persistence is associated with two single nucleotide
polymorphisms (SNPs) 5’ of LCT
-13910 C/T, -22018 G/A
Lactase has been under very
strong selection
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Perhaps?
Persistent
LCT
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Estimated age of lactase persistence haplotype 180 generations
(3,600 years)
Dairy farming: 5,000-9,000 years ago
Courtesy JN Hirschhorn Harvard
?Evolutionary cure of lactase deficiency
• Genetic signatures of recent positive
selection can be found
• LCT (lactase) shows remarkably strong
evidence of recent positive selection
• New metrics may locate other regions of the
genome that have been under recent
positive selection
Courtesy JN Hirschhorn Harvard
• December 2006: Reports of additional
mutations leading to persistence of lactase
in Africa in different dairy farming regions
• With signatures of strong positive selection
• ?Nature, Tischkof et al
Dietary Supplementation for Biotinidase deficiency:
Biotin Cycle
Symptom
>50%
Alopecia (hair loss)
Developmental delay
Hypotonia (poor muscle tone)
Ketolactic aciduria
Seizures
Skin rash/skin infection
2550%
Ataxia (poor coordination)
Conjunctivitis (redness of the eye)
Hearing loss
Lethargy (drowsiness)
Mild hyperammonemia
Breathing problems
Eye problems
1025%
Coma
Feeding difficulties/vomiting/diarrhea
Fungal infections
<10%
Hepatomegaly (enlarged liver)
Speech problems
Splenomegaly (enlarged spleen)
Effects of Dietary SupplementationTherapy
with Oral Pharmacologic Doses of Biotin
Before Biotin treatment
After Biotin treatment
Therapy of Genetic Disorders
• Gene Product Therapy
– Hormone, protein or enzyme replacement
• Hormone supplementation:
– Hypothyroidism: thyroid
– Congenital adrenal hyperplasia: cortisol
– Growth hormone
• Hemophilia; clotting factors
• Diabetes: insulin
• Enzyme replacement
– Beta glucosidase : Gauchers
– Alpha glucosidase: Pompe
– Adenosine deaminase (PEG): ADA- SCID
Enzyme Replacement Therapy of Inherited
Disorders
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Extension of paradigm of therapy for deficiency of
plasma proteins (eg hormones & clotting factors)
May not require targeting intracellularly if stored
or toxic metabolite is in equilibrium with plasma
Targeting of deficient protein into cells and
organelles would provide the widest application
Examples of Current Enzyme
Therapy
• Current FDA approved enzyme replacement
therapy
– Adenosine deaminase deficiency (SCID)• Severe combined Immunodeficiency
• No targeting to cells, but removal of metabolites from
plasma
– Several Lysosomal Storage Disorders
• Genetic deficiency of Lysosomal Enzymes
• Therapy: Targeting of deficient enzyme to lysosomes
Adenosine deaminase deficiency: cellular &
metabolic interactions
lymphoid cells &
RBCs
other cells
dATP nl
dATP
deoxyadenosine
Adenosine deaminase deficiency: cellular &
metabolic interactions:
Effect of enzyme therapy
lymphoid cells & RBCs
dATP
deoxy adenosine
Injection of PEG Calf
Adenosine Deaminase
deoxyinosine
Lysosomal Storage Diseases
• Lysosomes: intracellular organelles containing
hydrolytic enzymes that degrade macromolecules
(recycling and “garbage disposal” for cells)
• Lysosomal enzymes are targeted to lysosomes by
interaction with Mannose 6 PO4 receptors in the
cell
• Lysosomal enzymes can be taken up into the cell
from plasma by interaction with Mannose 6 PO4
receptors on cell surface
ENZYME REPLACEMENT THERAPY
FOR LYSOSOMAL STORAGE DISEASES
Disease
Current Status
• Gaucher Disease
Approved 1991
• Fabry Disease
Approved 2001/03
• Mucopolysaccharidosis I
Approved 2003
• Mucopolysaccharidosis VI
Approved 2005
• Mucopolysaccharidosis II
Approved 2006
• Pompe Disease
Approved 2006
• Niemann-Pick B Disease
Phase 1 Trial
Therapy of Genetic Disorders
• Cell or Organ Transplantation
Cells
Bone marrow
Immunodeficiency Disorders
Organs
Kidney
Fabry Disease
Liver
Tyrosinemia
Gene Therapy: Types
• Introduction of normal gene
– Somatic
– Germ line
• Therapy of noninherited disorders
– (cancer, AIDS)
• Production of gene product for
administration
– (hemophilia, growth hormone,
erythropoietin)
Somatic Gene Therapy
Introduction of recombinant genes into somatic
cells to treat genetic or acquired disease
• Does not involve germ line
• applicable to any disease with known
molecular basis of pathogenesis
• currently does not involve removal, repair or
site-specific replacement of mutant genes
• may not require permanent alteration of
cells (repetitive therapy)
Disease Characteristics Currently
Ideal for Gene Therapy
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Lethal disorder
Course not highly variable
Reversible
No universal therapy
Gene cloned
No tissue specificity or regulation
Bone marrow cells involved
State of the Art of Genetic
Engineering
• Ideal
– Replace defective gene with normal (site specific
insertion)
– Target vector containing the gene to damaged cell
– In vivo administration safe, effective and permanent
(integration into DNA but not at oncogenic sites)
– Vector contains all regulatory elements
• Current
– Site specific insertion very early and experimental
– No current trial incorporates all of the ideal
requirements
Gene Therapy Potential
Successes
Disease
Cell/tissue
Vector
X-linked & ADASevere Combined
Immunodeficiency
Stem Cell
(bone marrow)
Retrovirus
Somatic Mosaicism:
Reversion of an Inherited Mutation
to Normal and Selective Growth Advantage
Mutant
Reversion of mutation
to normal
Mutant cells
Normal cells
“Successful” Gene Therapy for
Immunodeficiency
Diseases:2005
• Retroviral vector used despite major disadvantages
• Over 14 patients with X linked severe combined
immunodeficiency of 3 different types have been
treated successfully
• Oncogenic insertion in two of 14 children-leukemia
• - X-linked SCID trials suspended but now
reinstituted
• ~8 patients with ADA deficiency treated
Current Therapy of Genetic
Disorders
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Preventive
Metabolic Manipulation
Gene Product Replacement
Cell or Organ Replacement
Gene Therapy
Current Therapy of Genetic
Disorders
• Experimental
• Gene Therapy (Experimental)
• Correction with oligonucleotide or
RNAi
• Silencing (RNAi and others)
• Transplicing
• “Read through” of nonsense mutations
Ideal Viral Vectors
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Replication defective
Accommodates large inserts
High titer with broad cell range
High level of expression of inserted gene
Unique promotors
– Tissue specific vs universal
– On/off switch; controllable expression
• Non-toxic
Current Enzyme Therapy of
Lysosomal Disorders with Intracellular Replacement
of Enzyme:
Currently “standard of care”
Gauchers Disease (beta glucosidase; non neuronopathic)
Current Clinical Trials:
Glycogen Storage Disease Type II (acid maltase)
Fabry Disease (alpha galactosidase)
Hurler Disease (alpha iduronidase)
Hunter Disease (iduronate sulfatase)
Lysosomes
Lysosomal enzymes contain a recognition site for
targeted uptake into cells and lysosomes
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enzyme protein synthesized in endoplasmic reticulum
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carbohydrate (high mannose) added in ER
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mannose 6 phosphate added in Golgi:
some enzyme secreted to outside of cell
mannose 6 phosphate binds to receptors, leading to
targeting to lysosomes and uptake of enzyme
into cells
Enzyme Replacement Therapy
for Lysosomal Disorders
• Requirements
– Unprocessed enzyme that will be taken up by cells and
targeted to lysosomes
• Solutions
– Purify from cultured cells
• (introduce gene into cells, amplify copy number, mass culture,
purify protein) Currently FDA approved approach
– Purify from milk of an animal
• (attach casein promotor to gene, introduce into ovum
(transgenic), select animal(s) producing greatest amount of
enzyme, purify enzyme from milk) Attractive but problems and
difficult to get approval
Considerations for Gene Therapy
• State of the art of genetic engineering
• State of the art of manipulation of cells and
organs
• Disease characteristics
Variables in Current Gene
Therapy Trials
• Vector for delivery of gene
• Ex vivo vs In vivo administration
• Permanent integration into DNA vs transient
expression
• Incorporation of regulatory elements
Examples of Current Enzyme
Therapy
• Current FDA approved enzyme replacement
therapy
– Adenosine deaminase deficiency (SCID)• Severe combined Immunodeficiency
• No targeting to cells, but removal of metabolites from
plasma
– Gaucher Disease (Beta glucosidase deficiency)
• Gaucher Disease (non-neuronal only)
• Lysosomal Storage Disorder
• Targeting of deficient enzyme to lysosomes
Types of Somatic Gene
Transfer
• Ex vivo
– Gene or expression vector carrying the gene
is inserted into explanted or cultured cells
which are then transplanted into the patient
• In vivo
– Gene or expression vector carrying the gene
is administered directly to the patient
ENZYME REPLACEMENT THERAPY
FOR LYSOSOMAL STORAGE DISEASES
Gaucher Disease
Fabry Disease
Approved 1991
Approved 2001 (EU), 2003 (US)
Mucopolysaccharidosis I Approved 2003 (EU & US)
Mucopolysaccharidosis VI Approved, 2005 (US& EU)
Mucopolysaccharidosis II Approved, 2006 (US)
Pompe Disease (AMD)
Niemann-Pick B Disease
Approved, 2006 (US & EU)
Phase 1 Trial Underway