1. Current Therapy of Genetic Disorders Preventive Metabolic Manipulation Gene Product Replacement Cell or Organ Transplantation Gene Therapy

2. 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

3. 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,)

4. 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

5. 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

6. ? 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

7. 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

8. Dietary Supplementation for Biotinidase deficiency: Biotin Cycle

9. 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 loss Lethargy (drowsiness) Mild hyperammonemia Breathing problems Eye problems 25- 50% Alopecia (hair loss ) Developmental delay Hypotonia (poor muscle tone) Ketolactic aciduria Seizures Skin rash/skin infection >50% Symptom

10. Before Biotin treatment After Biotin treatment Effects of Dietary SupplementationTherapy with Oral Pharmacologic Doses of Biotin

11. 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

12. 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

13. 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

14. Adenosine deaminase deficiency: cellular & metabolic interactions dATP dATP nl deoxyadenosine lymphoid cells & RBCs other cells

15. Adenosine deaminase deficiency: cellular & metabolic interactions: Effect of enzyme therapy dATP deoxy adenosine lymphoid cells & RBCs deoxyinosine Injection of PEG Calf Adenosine Deaminase

16. 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 PO 4 receptors in the cell Lysosomal enzymes can be taken up into the cell from plasma by interaction with Mannose 6 PO 4 receptors on cell surface

17. Gaucher DiseaseApproved 1991 Fabry DiseaseApproved 2001/03 Mucopolysaccharidosis I Approved 2003 Mucopolysaccharidosis VIApproved 2005 Mucopolysaccharidosis II Approved 2006 Pompe DiseaseApproved 2006 Niemann-Pick B DiseasePhase 1 Trial ENZYME REPLACEMENT THERAPY FOR LYSOSOMAL STORAGE DISEASES Disease Current Status

18. Therapy of Genetic Disorders Cell or Organ Transplantation Cells Bone marrowImmunodeficiency Disorders Organs KidneyFabry Disease LiverTyrosinemia

19. 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)

20. 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)

21. 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

22. 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

23. Gene Therapy Potential Successes DiseaseCell/tissueVector X-linked & ADA-Stem CellRetrovirus Severe Combined (bone marrow) Immunodeficiency

24. Mutant Somatic Mosaicism: Reversion of an Inherited Mutation to Normal and Selective Growth Advantage Reversion of mutation to normal Mutant cells Normal cells

25. “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

26. Current Therapy of Genetic Disorders Preventive Metabolic Manipulation Gene Product Replacement Cell or Organ Replacement Gene Therapy

27. Current Therapy of Genetic Disorders Experimental Gene Therapy (Experimental) Correction with oligonucleotide or RNAi Silencing (RNAi and others) Transplicing “Read through” of nonsense mutations

29. 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

30. 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)

31. Lysosomes

32. 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

33. 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

34. Considerations for Gene Therapy State of the art of genetic engineering State of the art of manipulation of cells and organs Disease characteristics

35. 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

36. 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

37. 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

38. Gaucher DiseaseApproved 1991 Fabry DiseaseApproved 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 DiseasePhase 1 Trial Underway ENZYME REPLACEMENT THERAPY FOR LYSOSOMAL STORAGE DISEASES