GENE THERAPY

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GENE THERAPY

• ??. ????? ????????????????
• ??. ?????? ????????????????
• ??. ?????? ?????????

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????????????? GENE THERAPY

• 1964 sequence ??????????? DNA
  ?????????????????
• ?????????????????????????????
  ??
• 1978 clone ???????????????? ------- Human
  insulin
• 1986 ?????? Human hepatitis B vacine
  ??????? genetic
• engineering
• 1988 ?????????????????????????????
  ????????????????
• ????????????

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??????????????????????????????

• ??????????????????
  ???????
• ?????????????
• ???????????????????? Down s syndrome
• ??????????????? Leukemia
• ????? DNA
• ??????????????? Thalassemia
• ???????????????? Haemophelia

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????????????????????????????????? DNA
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??????????????????????????????????????

• ?????????????
• ??????????????????? ??????????????????????????
  ?????????????? ???? ???????????? ????????????
  ? ???????????? ??????????????????? ???????
• ????????? Gene Therapy
• ???????????????????????? ?????????????????????
  ?? ???? ??????? ???????????????
  ????????????? ?????????? ???????
  ???????????????????????????????????????????????
  ???????????????????????

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?????????? DNA ?? genome

• Codon ?????????????????????????????????????
• AAA lysine UUU phenylalanine
• RNA ????? 4 ??? ( A,U,C,T ) ?????????????????????
  ??????
• 43 64 codon
• - ????????????????????? 20 ????
• - start signal AUG ( methionine )
• - stop signal UAA , UAG , UGA

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?????????????????????????????
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?????????????
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??? isolate ???

• Peptide mapping
• ????? oligonucleotide primer
• ????????? RT-PCR ??????????????? DNA
• ??? DNA ???????????????? restriction enzyme
  ????????
• ??????????????????????? electrophoresis
• ????????? sequence ????????????????

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?????????????????????? DNA

• restriction endonuclease enzyme
• restriction endonuclease I
  restriction endonuclease II
• -CCGC-
  -TTAA-
• -GGCG-
  -AATT-
• 
  
• -CCG C-
  -TTA A-
• -GGC G-
  -AAT T-

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• 
  -ATCCCGCCTTAACC-
• 
  -TAGGGCGGAATTGG-
• RT I
  RT II
• -ATCCCG CCTTAACC -ATCCCGCCTTA
  ACC-
• -TAGGGC GGAATTGG -TAGGGCGGAAT
  TGG

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???????????????????????????????????????
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????????????????????????????

• ?????? recombinant DNA
• - plasmid
• - bacteriophage
• ?????? clone ?????? retrovirus

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?????? recombinant DNA
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????????? clone ????????? retrovirus
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Gene Therapy

• What is gene therapy?
• novel approach to treating diseases
• base on modifying the expression of a
  persons genes toward a therapeutic goal

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Procedure of transferring genetic material

• DNA chain
• cut DNA chain
• separate the selected pieces of DNA
• therapeutic DNA sequence
• placed in the DNA of vector
• insert the vector into target cells

Restriction enzyme
electrophoresis
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Gene Therapy

• Somantic gene therapy
• ex vivo
• in vivo
• Germline gene therapy
• treat pre-embryo requires vitro fertilization
• treat germ cells ( sperm or egg cells)

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vectors

• Viral vector
• lytic viral vectors
• retroviral vectors
• parvoviral vectors
• nonviral vector
• lipid encapsulation facilitate passage across
  cell membrane
• oligos oligonucleotides with about 25 bases ,
  to be hooked up to tiny gold bead which can shot
  into skin
• electroporation
• biolistic using high velocity tungsten
  microprojectiles to insert DNA

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Four thing to choosing type of viral vectors

• When choosing which type of virus to use for gene
  therapy
• ease with which the virus can be
  handled,manipulate, grown to high titers in vitro
• the efficiency with which virus can infect the
  target cells
• the specificity, a virus has for certain types of
  cells
• the virus can evade the immune system and not
  induce an inflammation response

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Lytic viral vectors

• Lytic viral vectors adenovirus, herpes simplex
• adventage
• grown in vitro to high titers
• easy to handle and easy for vector construction
• vector preperation is low cost
• disadventage
• therapeutic gene to be active for only a short
  time ( do not integrate into host cell genome)
• if repeat exposure ,the immune will remember
  antigen , induce inflammatory response

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Retroviral vectors

• simple retroviral vectors
• moloney murine leukemia virus
• require a dividing target cells
• complex retroviral vectors
• Human foamy virus, human immunodeficiency virus
• can integrate non dividing target cells

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Parvoviral vectors

• Similar to retroviral vectors
• can integrate into chromosomal DNA
• ability to transduce non dividing target cells (
  in a variety of tissues)
• not associated with any diseases in human
• ex adeno associated virus (AAV)

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Initial studies

• 1990-1991 first successful treatment of adenosine
  deaminase deficiency (ADA)
• the absence of ADA result in premature
  death of T lymphocytes, leading to severe
  combined immunodeficiency (SCID)
• treat transferred a normal ADA gene
  into lymphocytes
• put ADA gene into bone
  marrow
• both strategies was ex vivo

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Initial studies

• 1992 successful treatment of familial
  hypercholesterolemia
• patients lack the hepatic receptor
  that clears low - density lipoprotein (LDL) from
  blood . In the absence of the receptor , patient
  exhibit extreme hyperlipidemia
• treat ex vivo hepatocyte from
  patient ,exposure to a virus carring the human
  LDL receptor gene. The cells were infused into
  patient s portal vein.

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Gene Therapy and Patient Care

• ???????????????? Gene Therapy
• The Replacement of a nonfunctional protein to
  restore a normal cell function
• The modulation of enzyme activity to increase
  therapeutic efficacy
• The stimulation of the immune respond by
  augmenting antibody production
• The detoxification of deterious agent

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The replacement of a nonfunctional protein

• ???????????????? nonfunctional protein
  ???????? normal cell ????????????? ??????????
  gene mutation ?????????????????????????? amino
  acid sequence ????? protein ??????????????????????
  ?? ?????????? ??? gene therapy
  ????????????????????
• ????????
• ???????hemophilia ?????????????? specific
  clotting protein ????????? bleeding ??? Gene
  therapy ?????????????? Clotting Protein
• Sickel Cell anemia ??????????? cloning gene

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The modulation of enzyme activity

• Gene Therapy ????????????????????????? ????????
  metabolism ????? ???????????? activity ??? enzyme
  ???? gene therapy
• 5 - fluorouracil uridine phosphorylase
• ?????????????????????????????????????????? enzyme
  ??????????????? Catabolism ???? ??????
  allopurinol ???????????????????? ?????? inhibit
  xanthine oxidase ??????????????????????
  ???????????? ??? gene ?????????????????? enzyme
  ???? ??????????????? mutation

5 fluorodeoxyuridine nomophosphate
Uridine kinase
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The stimulation of the immune response

• Gene therapy ????????????? antibody ?????????
  ??????????? Cell circulation ??????????????????
  lymphocytes

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The detoxification

• ??????????????????????? ?????????????? ( stress )
  ?????????????????????? oxidation
  ?????????????????????????????????????? Gene
  Therapy ?????????? antioxidant capacity ??????
  introduction ??? gene ???????????????????
  internal antioxidant

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??????????????? Gene therapy ??????????

• 1.Alzheimers Disease
• 2.Cancer
• 3.Diabetes
• 4.Hemophilia

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Gene therapy and Alzhiemer s Disease

• Utility of the Apolipoprotein E Genotype
  ?????????????????????????????????? alzheimer

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Gene therapy and Alzhiemers Disease ( Continue)

• First gene therapy experiment for Alzheimers
• 10 ?????? 2544 ?????????????????????????????
  ?????????????? Gene therapy ?????????????? 60 ??
  ?????????????????????????????????????????????
  ????? 3 ?????? ???????????????????????????????????
  ????????????? ???????????????????? human growth
  factor ???????????????????????????????????
  frontal lobe
• ?????????????????????? ???????
• nerve growth factor ????????????????
  Cell??????????
• Cell ?????????????????????????????????????????????
  ?????????????
• ??????????????????????????????????? ????? 12
  ?????? ????? nerve growth factor
  ?????????????????????? cholinergic cells ????????
  aging cell ?????????? ?????????????????????????

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Gene therapy and Cancer

• Gene therapy shows promise against cancer
• Dr.Fadlo Khuri at the university of Texus
  August 4,2000
• ???????????? 30 ????????? Head and neck cancers
• ??????????? genetically engineered adenovirus (
  ONYX - 015 ) ??????? Standard chemotherapy Result
• ??????? 19 ????????? tumor ??????????????????????
  ????? ????? 8 ?????? Tumor ????????????

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Gene therapy and Diabetes

• ??????????? ??????????????????? 8 ??
  ???????????????? Insulin ?????????????????????????
  ????? 1 ?? ?????????????????????????? Insulin ???
  glucagon ???
• ??????????? islet cell ??????????????????????
  ?????????? Cell ??? Donor ?????????
• ?????????????????????????????????????
  cellmultiplying gene ?????????? human islet cells
  ????????????????????????? ? ?????

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Problem associated with Gene Therapy

• Delivery system for gene therapy is not perfect.
• Pharmacokinetics of the synthesized product is
  unknown what mechanism exist with the body to
  transport, activate,or degrade the particular
  product.
• Need to be transfected the appropriate cells in
  order to achieve therapeutic efficiency.
• Impact on treatment because of relating to
  alteration of the genetic composition of human
  being.

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Good bye