Current Therapy of Genetic Disorders

• 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 pillsAll other mammals and most people lose the ability to digest lactose by adulthoodLactase persistence is found in 50-90% of Europeans but is much rarer in other populationsLactase 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

Persistent LCT

+ Perhaps?

????

√

Courtesy JN Hirschhorn Harvard

Estimated age of lactase persistence haplotype 180 generations (3,600 years)

Dairy farming: 5,000-9,000 years ago

?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

Hepatomegaly (enlarged liver)Speech problems Splenomegaly (enlarged spleen)

<10%

Coma Feeding difficulties/vomiting/diarrhea Fungal infections

10-25%

Ataxia (poor coordination)Conjunctivitis (redness of the eye)Hearing lossLethargy (drowsiness)Mild hyperammonemiaBreathing problemsEye problems

25-50%

Alopecia (hair loss) Developmental delay Hypotonia (poor muscle tone)Ketolactic aciduriaSeizuresSkin rash/skin infection

>50%

Symptom

Before Biotin treatment After Biotin treatment

Effects of Dietary SupplementationTherapy with Oral Pharmacologic Doses of Biotin

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

• Extension of paradigm of therapy for deficiency of plasma proteins (eg hormones & clotting factors)

• May not require targeting intracellularly if stored

• Targeting of deficient protein into cells and organelles would provide the widest application

or toxic metabolite is in equilibrium with plasma

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

dATP dATP nl

deoxyadenosine

lymphoid cells & RBCs

other cells

Adenosine deaminase deficiency: cellular & metabolic interactions:

Effect of enzyme therapy

dATP

deoxy adenosine

lymphoid cells & RBCs

deoxyinosine

Injection of PEG Calf Adenosine Deaminase

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

• 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

ENZYME REPLACEMENT THERAPY

FOR LYSOSOMAL STORAGE DISEASES

Disease Current Status

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 TherapyIntroduction 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

• 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 & ADA- Stem Cell Retrovirus

Severe Combined (bone marrow)

Immunodeficiency

Mutant

Somatic Mosaicism: Reversion of an Inherited Mutation

to Normal and Selective Growth Advantage

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

• 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• 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

• enzyme protein synthesized in endoplasmic reticulum

• carbohydrate (high mannose) added in ER

• 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

Lysosomal enzymes contain a recognition site for targeted uptake into cells and lysosomes

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

Gaucher Disease Approved 1991

Fabry Disease 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) Approved, 2006 (US & EU)

Niemann-Pick B Disease Phase 1 Trial Underway

ENZYME REPLACEMENT THERAPY FOR LYSOSOMAL STORAGE DISEASES