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Therapy of Genetic Disorders MGL-14 May. 6 th 2015

Therapy of Genetic Disorders MGL-14 May. 6 th 2015. Mohammed El-Khateeb. GENE THERAPY. Replacement Therapy Gene transfer Gene manipulation Cloning Stem cell. GENE. Gene Product. Metabolic Functional Structural Effect Effect Effect.

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Therapy of Genetic Disorders MGL-14 May. 6 th 2015

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  1. Therapy of Genetic DisordersMGL-14May. 6th 2015 Mohammed El-Khateeb 台大農藝系 遺傳學 601 20000

  2. GENE THERAPY Replacement Therapy Gene transfer Gene manipulation Cloning Stem cell

  3. GENE Gene Product Metabolic Functional Structural EffectEffect Effect

  4. 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

  5. Gene Therapy Strategies • Interference with gene products • Replacement of a missing or defective gene • Introduction of gene(s) to influence cellular process

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

  7. Gene Replacement strategy • Applies to diseases caused by single gene defects • Transfer of a functional copy of the defective or missing gene • Examples: enzyme deficiencies

  8. Gene Replacement Strategy • To apply this strategy, three requirements must be met: • The specific gene defect must be known • A functional copy of the gene must be available • Target cells must be available and amenable to transfection methods resulting in long-term expression

  9. 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

  10. Gene Replacement Strategy

  11. 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

  12. Gene Transfer: Types of Vectors • RNA viruses (Retroviruses) 1. Murine leukemia virus (MuLV) 2. Human immunodeficiency viruses (HIV) 3. Human T-cell lymphotropic viruses (HTLV) • DNA viruses 1. Adenoviruses 2. Adeno-associated viruses (AAV) 3. Herpes simplex virus (HSV) 4. Pox viruses • Non-viral vectors 1. Liposomes 2. Naked DNA 3. Liposome-polycation complexes 4. Peptide delivery systems

  13. 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

  14. 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

  15. Ex vivo gene therapy • The genetic material is first transferred • into the cells grown in vitro 2. Controlled process; Genetically altered cells are selectedand expanded; more manipulations 3. Cells are then returned back to the patient

  16. In vivo and ex vivo gene therapy concepts

  17. Proposed concept of designer nuclease‐mediated correction of patient‐specific iPSC for autologous transplantation.

  18. Gene therapy could be very different for different diseases Gene transplantation (to patient with gene deletion) Gene correction (To revert specific mutation in the gene of interest) Gene augmentation (to enhance expression of gene of interest)

  19. Barriers to successful gene therapy: • Vector development • Corrective gene construct • Proliferation and maintenance of target cells • Efficient transfection and transport of DNA to nucleus for integration into genome • Expansion of engineered cells and implantation into patient

  20. Creation of recombinant DNA molecules in vitro plasmid cloning vector

  21. SCID treatments Life in germ-free envinronment Bone-marrow transplantations Enzyme replacement therapy VERY expensive; not a cure; temporary effect GENE THERAPY

  22. “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

  23. SCNT: Somatic Cell Nuclear Transfer • SCNT is a method used for: • Reproductive cloning such as cloning an embryo • Regenerative cloning to produce “customized” stem cells & overcome immune rejection • Blastula stage cannot continue to develop in vitro • It must be implanted into surrogate mom • Surrogate mom is just a container that provides protection & chemical signals necessary for development

  24. Protein Production in Transgenic Sheep YFG=  Your Favorite Gene

  25. Spectrum of Gene expression Cancer Gene Therapy • Oncogene inactivation • Augmentation of TSG • Cell targeted suicide-pro-drug to toxic metabolite by transfer of converting enzyme gene into tumor cells • Chemoprotection - transfer of MDR ( Multi Drug Resistance) gene into bone marrow cells to decrease effect of cytotoxic agents

  26. Drug Activation Gene Therapy for Cancer

  27. Oral Manifestations of Genetic & Congenital Disorders

  28. Historically, there has been great synergy between genetics and oral medicine. Specifically, two extraordinary men have made major contributions to the field:RobertGorlin, D.D.S, M.S. (1924-2006) • M. Michael Cohen, Jr, D.D.S., Ph.D. (1937- )

  29. Syndromology: Definitions I • Malformation: an abnormality in form or function caused by an alteration in the tissue primordia (examples: cleft palate, dentinogenesisimperfecta) • • Deformation: an abnormality that results from unusual forces acting on normal tissue (example: facial asymmetry due to plagiocephaly caused by extra uterine positioning of the head) • • Disruption: an abnormality resulting from breakdown of normal tissue (example: amniotic band syndrome) • • Dysplasia: an abnormality resulting from abnormal organization of cells in tissue (example: neurocutaneousmelanosis sequence)

  30. Syndromology: Definitions II • Syndrome: A group of malformations, deformations and malformation sequences, etc. that occur together due to some identifiable underlying cause (examples: Down syndrome, Marfan syndrome, fetal alcohol syndrome) • • Malformation sequence: A group of malformations that arise as the result of a single underlying malformation (ie. Pierre Robin malformation sequence) • • Association: A group of malformations (etc.) that occur together more often than would be expected by chance, but in which no underlying etiology can be identified (example: VACTERL association)

  31. Review of Some Common Genetic and Congenital Disorders: Background • 2-3% of all newborns will be found to have one or more abnormalities in the newborn period. • By the age of 1 year, 6% of individuals will have been found to have an abnormality. • Geneticists classify these disorders into 5 categories: • Chromosomal abnormalities (7.5%) • Single gene disorders (7.5%) • Teratogenic disorders (6%) • Multifactorial disorders (40%) • Etiology unknown (40%)

  32. Chromosomal Abnormalities Turner syndrome: Oral & Dental • Premature eruption of the teeth • High arched palate • Increased molarization of premolars • Cusp and crown size are reduced • Prior to treatment, may need prophylactic antibiotics (due to associated cardiac disease)

  33. Chromosomal Abnormalities Turner syndrome: Oral & Dental • MaIocclusion • Anodontia (missing teeth) • Malformed teeth (microdontia) • Enamel hypoplasia • Poor dental hygiene with cavities and gum disease

  34. Chromosomal Abnormalities Williams Syndrome: Oral and Dental • MaIocclusion • Anodontia (missing teeth) • Malformed teeth (microdontia) • Enamel hypoplasia • Poor dental hygiene with cavities and gum disease

  35. Single Gene Disorders • Autosomal dominant inheritance (osteogenesis imperfecta) • Autosomal Recessive inheritance (Ellis-van Creveld syndrome) • X-linked disorders (hypohidrotic ectodermal dysplasia, Incontinentia pigmenti)

  36. Single Gene Disorders OI type I: Oral and Dental • Problems include: • Dental fractures • Premature “wearing down” of teeth • Cosmetic issue • Delayed dental eruption • Treatment: Goal is to maintain functional occlusion, optimal gingival health, and overall appearance.

  37. Single Gene Disorders Autosomal Recessive Disorders: Ellis-van Creveld Syndrome (EVCS) • AR Disorders occur when an offspring inherits two copies of a non-working gene from parents • EVCS is a rare AR disorder (prevalence=1 in 60,000) • Much more common in the old order Amish population (“founder effect”) • Caused by mutations in the EVC and EVC2 genes (function unknown)

  38. Ellis-van Creveld Syndrome (EVCS) Oral and Dental Features • Neonatal teeth • Partial anodontia • Small teeth • Delayed eruption • Thickened oral frenula, with upper lip bound to alveolar ridge

  39. X-Linked Disorders Hypohidrotic Ectodermal Dysplasia (HED) • X-linked disorders are caused by mutations on the X chromosome • HED is an X-linked recessive disorder, passed from carrier mothers to affected sons • Occurs in 1 in 10,000 newborns (all boys) • Caused by a mutation in the EDA gene (gene product = Ectodysplasin-A)

  40. HED: Oral and Dental I • May develop only 5 to 7 teeth (canines and 1st molar) • Teeth are small with conical crowns. • Paucity of saliva (thick) • Carrier females may have minor dental anomalies • Dental treatment must begin at an early age. • Bonding of conical shaped teeth in young individuals improves esthetics and chewing ability. • Orthodontics may be necessary.

  41. HED: Oral and Dental II • Dental implants in the anterior portion of the mandible are only successful in children >7 y.o. • Prostheses may need to be replaced every 2.5 yrs. • Because of problems with chewing and swallowing, dietary counseling may be helpful

  42. Crouzon Syndrome • AD inheritance (mutation in FGFR2 or FGFR3) • Prevalence= 1 in 20,000 • Clinical features: • Abnormal skull shape (depending on involved sutures) • Facial: Ocular hypertelorism, proptosis, midface hypoplasia, beaked nose, and prognathism • Obstructive apnea • Other: nl intelligence, and extremities, hydrocephalus, increased ICP

  43. Crouzon Syndrome: Dental & Orthodontic • Mandibular prognathism with midface hypoplasia • V-shaped maxillary arch • Overcrowding of upper teeth with malocclusions • Narrow, high palate (occasionally cleft) • Occasional oligodontia, macrodontia, peg-shaped, and widely spaced teeth • The pediatric dentist & orthodontist should function as part of a multidisciplinary team in planning care of patients with this & other craniosynostosis syndromes.

  44. Treacher Collins syndrome (TCS) • AD inheritance (mutation in TCOF1 gene) • Prevalence = 1 in 10,000 to 1 in 50,000 • Clinical features (variable expression):Symmetric facial anomalies: micrognathia with extreme shortening of mandible; colobomata of lower eyelid; microtia, macrostomia • Respiratory: Severe obstructive apnea (due to PRS, choanal atresia/stenosis) • Usually normal intelligence

  45. TCS: Dental & Orthodontic Implications • Dental anomalies occur in 60%, with 1 to 8 per individual tooth agenesis (33.3%), • enamel opacities (20%), • ectopic eruption of the maxillary first molars (13.3%) • Less frequently observed features: Nasal deformity • High-arched palate • Angle class II anterior open-bite malocclusion

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