Chapter 1 slide 1

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Therapy of Genetic DisordersMGL-14May. 6th
2015
Mohammed El-Khateeb
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GENE THERAPY

• Replacement Therapy
• Gene transfer
• Gene manipulation
• Cloning
• Stem cell

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GENE
Gene Product
Metabolic Functional Structural Effect
Effect Effect
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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

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Gene Therapy Strategies

• Interference with gene products
• Replacement of a missing or defective gene
• Introduction of gene(s) to influence cellular
  process

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Considerations for Gene Therapy

• State of the art of genetic engineering
• State of the art of manipulation of cells and
  organs
• Disease characteristics

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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

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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

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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

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Gene Replacement Strategy
Gene with defect Disease/Disorder
Adenosine deaminase (ADA) SCID
a-1-antitrypsin Emphysema
CF transmembrane regulator Cystic fibrosis
Clotting factor VIII Hemophilia A
Clotting factor IX Hemophilia B
b-chain of hemoglobin Sickle cell anemia
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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

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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

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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

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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

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Ex vivo gene therapy

• The genetic material is first transferred
• into the cells grown in vitro

2. Controlled process Genetically altered
cells are selected and expanded more
manipulations
3. Cells are then returned back to the patient
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In vivo and ex vivo gene therapy concepts
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Proposed concept of designer nuclease-mediated
correction of patient-specific iPSC for
autologous transplantation.
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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)
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Barriers to successful gene therapy

1. Vector development
2. Corrective gene construct
3. Proliferation and maintenance of target cells
4. Efficient transfection and transport of DNA to
   nucleus for integration into genome
5. Expansion of engineered cells and implantation
   into patient

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Creation of recombinant DNA molecules in vitro
plasmid cloning vector
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SCID treatments
Life in germ-free envinronment
Bone-marrow transplantations
Enzyme replacement therapy VERY expensive not a
cure temporary effect
GENE THERAPY
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Successful Gene Therapy for Immunodeficiency
Diseases2005

• 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

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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

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Protein Production in Transgenic Sheep
YFG  Your Favorite Gene
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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

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Drug Activation Gene Therapy for Cancer
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Oral Manifestations of Genetic Congenital
Disorders
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• Historically, there has been great synergy
  between genetics and oral medicine. Specifically,
  two extraordinary men have made major
  contributions to the fieldRobert Gorlin, D.D.S,
  M.S. (1924-2006)
• M. Michael Cohen, Jr, D.D.S., Ph.D. (1937- )

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Syndromology Definitions I

• Malformation an abnormality in form or function
  caused by an alteration in the tissue primordia
  (examples cleft palate, dentinogenesis
  imperfecta)
• 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 neurocutaneous melanosis sequence)

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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)

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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)

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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)

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Chromosomal Abnormalities Turner syndrome Oral
Dental

• MaIocclusion
• Anodontia (missing teeth)
• Malformed teeth (microdontia)
• Enamel hypoplasia
• Poor dental hygiene with cavities and gum disease

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Chromosomal Abnormalities Williams Syndrome Oral
and Dental

• MaIocclusion
• Anodontia (missing teeth)
• Malformed teeth (microdontia)
• Enamel hypoplasia
• Poor dental hygiene with cavities and gum disease

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Single Gene Disorders

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

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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.

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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 (prevalence1 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)

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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

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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)

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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.

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HED Oral and Dental II

• Dental implants in the anterior portion of the
  mandible are only successful in children gt7 y.o.
• Prostheses may need to be replaced every 2.5 yrs.
  
• Because of problems with chewing and swallowing,
  dietary counseling may be helpful

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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

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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.

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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

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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