gene therapy

1
GENE THERAPY
???????? ????????????????????????????????????????
??? ?????? ?????????????/??????????????
????????????????????????????????????
?????????????????????????????????? ???????????????
???????/???????
2
????????????????????? candidate diseases
1. Classical genetic diseases e.g. Inborn
error of metabolism
2. Complex genetic diseases (multifactorial
disorders) ???????? ??????? ??????
3. Viral infectious diseases ???????????????
(virus) ????????????/???????????
????????????????
3
Ethical Issues
Germ Cell Therapy (Manipulation of ovum and sperm)
In vitro fertilization Therapy Enhancement
Somatic Cell Genome Manipulation (Insertion of
genes using a vector)
Therapy Enhancement
Tissue replacement by stem cells (Embryo,
autologous progenitor or adult cells)
4
Eugenics is a term presented by Galton
good genes
This philosophy deals with all influences
that improve the inborn qualities of a race
either by enhancing good attributes (positive
eugenics) of an individual or by removing
unwanted attributes (negative eugenics).
5
Negative Eugenics
6
Positive Eugenics
7
Recent Eugenics History
1911, 1921, 1932 Three International Eugenics
Congresses participants such as Alexander Bell,
Major Leonard Darwin (son of Charles), William
Osler, Winston Churchill, Charles Eliot
(president of Harvard). These conferences were
abandoned.
1927 Buck v Bell gives Supreme Court approval of
castration laws. 1959 - 1990 Singapore Prime
Minister Lee Kuan Yew instituted positive and
negative eugenics Women who went to college were
encouraged to marry and rewarded with housing
and money. Women who had fewer than 2 children
and only a high school education were induced
by money to be sterilized. 1970s-present China
encourages one child families for reasons of
food supply but female infanticide is practiced
to assure male offspring to work the land. 1994
- China Maternal and Infant Health Care Law
requires pre-marital check-ups and gives to
doctors not patients the decision to abort.. 90
of Chinese geneticists approve and only 5 of
American approve.
8
Short History of Gene Manipulation Technology
In vitro fertilization (1978 to present)

• Somatic gene therapy in human patients (1990)
• Preimplantation selection
• Cloning mammals (1997)

9
(No Transcript)
10
(No Transcript)
11
(No Transcript)
12
(No Transcript)
13
Fate of Embryos at IVF Clinics

• Since 1978 over 100,000 embryos have been
  destroyed at IVF clinics.
• Currently there could be 1,000,000 embryos in
  storage in the United States (e.g. at UCSF clinic
  3,300 are in storage)
• Survey of 239 IVF clinics in the United States
  reveals
• 27 clinics donate to diagnostic tests mostly
  with the mothers permission
• 55 donate embryos to research
• 115 immediately discard unused embryos

14
Sources of Stem Cells
Uncontroversial Sources
Umbilical Cords
These are currently desired for many uses
Cord blood is frequently not stored
Adult Stem Cells
These do not seem as clinically effective
Controversial Sources
Aborted Fetuses
Frozen Spare Embryos
Cloned Human Embryo
Sources That Should Not be Controversial
Designed Stem Cell
From Modified Female Egg
15
(No Transcript)
16
Somatic Cell Gene TherapyTransfection
September 1990 - First sanctioned human study on
4 year old girl with adenosine deaminase
deficiency which leaves patient with a
compromised immune system (B and T cells). This
is one of the severe combined immune deficiencies
(SCID). A second patient is treated shortly
thereafter. Both improved but were not cured.
Though there is a wealth of animal data and
extensive peer review of the protocol presented
for this study, a particular human event which
occurred in 1984 did much in my view to move
ahead the concept of infusing viral vectors with
new genes. This is the episode of the bubble boy
which started when David was found to have SCID
in 1971 from which his brother had died 10 months
earlier. After a gap of another 16 years
success in SCID was reported in 4 subjects (April
2002, NEJM).
17
Gene Therapy for Parkinsons Disease
Monkey Brain Study
Now 24 mos
18
Monitoring Gene Therapy
19
(No Transcript)
20
Buying, Selling, Appropriating, Stealing and
Donating Human Body Organs and Parts
Ethical Considerations
Who owns an individuals blood cells taken as
part of a routine clinical exam?
Who owns the medical histories of a population
taken to advance medical science?
What is unfair about buying a kidney or other
dispensable body part?
Is there anything unethical or illegal about
stealing a body once in a grave? What about a
mummy?
Who should receive a heart or kidney if there are
not enough to go around?
When we do have an artificial heart in
production, how do we regulate?
21
Parthenogenesis
Parthenotes
Embryos grown from unfertilized eggs that, in
mammals, are not capable of developing into
viable fetuses
Experiment
77 primate (non-human) ova are stimulated by
pregnant horse serum
(Cibelli, Science 2002)
28/77 eggs reached metaphase II and were
parthenogenetically activated
4/28 eggs developed to blastocyst stage
1 stable cell line was obtained which could be
extensively propagated in vitro (10 months) while
maintaining their undifferentiated state
Cell Line
Cardiac myocytes
Smooth Muscle Cells
Beating ciliated epithelium
Adipocytes
Neurons
Dopaminergic
Serotonergic
22
Federal Ethical Guidelines
The Belmont Report 1979
Now 22 years old
The principles are still sound, however, current
extrapolations and recommended implementations
are controversial
Basic Principles
Autonomy - self governing / self directed
Beneficence - doing or producing good
Justice - equal burden / equal benefit
e.g. Do no harm, maximize benefits, minimize risk
No guidance for
Intergenerational risks
Stem cell uses
23
Informed Consent Knowledge of and consent to a
particular form of treatment and experiment
Four Elements
Three Standards

• Make a decision
• Select a choice
• Selected choice is reasonable

1. Competence 2. Disclosure 3.
Comprehension 4. Voluntariness

• What others would tell patient?
• What a reasonable person would want to know?
• What would this patient really want to know?

• Fully informed vs. adequately informed
• Translation to what the patient can comprehend
• Varies with patient

• Free from undue pressure
• But we all have some pressures and they vary
      with the individual.

I made him an offer he could not refuse. The
Godfather
24
GENE THERAPY
???????? ????????????????????????????????????????
??? ?????? ?????????????/??????????????
????????????????????????????????????
?????????????????????????????????? ???????????????
???????/???????
25
????????????????????? candidate diseases
1. Classical genetic diseases e.g. Inborn
error of metabolism
2. Complex genetic diseases (multifactorial
disorders) ???????? ??????? ??????
3. Viral infectious diseases ???????????????
(virus) ????????????/???????????
????????????????
26
????????????????????????????????????????

• Severe combined- immune deficiency (SCID)
  ?Adenosine deaminase
• Gauchers disease ?Glucocerebrosidase
• Duchen muscular dystrophy (DMD) ?Dystrophin
• Sickle cell anemia ?Beta globin
• Thalassemia ?Beta globin
• Hemophilia A ?blood clotting factor
• Phenylketonuria (PKU) ?Phenylalanine
  hydroxylase
• Emphysema ?Alpha-1 antitrypsin
• Cystic fibrosis ?Cystic fibrosis transmembrane
  regulator
• Familial hypercholesterolemia ?LDL receptor

27
??????????????????????????????????????

• SCID ?bone marrow transplant, ?????????? ADA
• Gauchers disease ? ??????????, ???????,
  ?????????????, bone marrow transplant
• Duchen muscular dystrophy (DMD) ?????????????????
  ????
• Sickle cell anemia ?blood transfusion, bone
  marrow transplant
• Thalassemia ? blood transfusion, bone marrow
  transplant, ?????????????
• Hemophilia A ?????????????? factor VIII
  ????????????
• Phenylketonuria (PKU) ????????????????
  phenylalanine
• Emphysema ????????????
• Cystic fibrosis ? ?????????????, ???????????
• Familial hypercholesterol ? ???????????,
  ???????????

28
???????????????????????? ????????
???????????? ??????????????????? SCID
Adenosine deaminase Emphysema a-1
antitrypsin Cystic fibrosis CFTR Hemophilia
A Factor VIII Hemophilia B Factor
IX Thalassemia Beta-globin Sickle cell
anemia Beta-globin Familial hypercholesterolemia
LDL receptor Gauchers disease glucocerebrosida
se
29
Gene replacement therapy
???????????????????????????????
?????????????? ??? Recessive Autosomal
recessive, X-linked recessive ???????????
?????????
30
Autosomal recessive

• Need to be homozygous for allele
• parents are usually unaffected carriers
• affects either sex
• increased incidence of parental consanguinity
• affected child, 25 of subsequent children
  affected

31
Autosomal recessive

• Cystic fibrosis
• - 1 in 2,500 in northern Europe (1 in 25 carrier
  frequency)
• - screening of newborns, can screen for carriers
• - chronic lung disease
• - pancreatic insufficiency
• - median survival 25 years but variable clinical
  severity

32
X-linked recessive

• Affects almost exclusively males
• mother is normally asymptomatic carrier
• effects on offspring
• - carrier female - 50 chance of affected male,
  50 chance of carrier female
• - affected male - 100 carrier females, no
  affected males
• - females may be affected if an affected father
  and carrier mother

33
X-linked recessive

• Duchenne muscular dystrophy
• - onset in early childhood (90 lt 5 years)
• - progressive muscle weakness
• - death usually by 20 years
• - null mutation in dystrophin
• Becker muscular dystrophy
• - onset in late childhood
• - chairbound by 25 years after onset but may
  have normal lifespan
• - reduced amount or function of dystrophin

34
Gene augmentation
?????????????????????????????????????????????????
?? ?????? ???? ?????????? (???? ---gt ???????)
e.g. ?????????????????????????????? tissue
plasminogen activator
?????????????????????
???????? plasminogen
??????? Thrombosis
35
In vivo gene therapy
Cystic fibrosis
??? CFTR gene ?????? adenoviral vectors
expressing ????????????? (aerosal to the lung)
Animal model ??? In vivo gene therapy
hemophilic dog ??? clotting factor factor
IX ??? adenoviral vector
36
Ex vivo gene therapy ???????? ??? SCID
Adenine deaminase (ADA) deficiency
(Autosomal recessive chromosome 20)
enzyme ????? purine metabolism
ADA
Metabolites ???? ???????? T-cell
immune deficiency (??????????????) (??????????????
?????)
37
Ex vivo gene therapy matched bone marrow
transplant
???? T-cell ??? ?.?. ADA def. 2 ?? ??????????
tissue culture dish with a retroviral vect
or directing ADA gene expression
ADA enzyme injection
Improved follow up (6 yr)
38
Ex vivo gene therapy Familial hypercholesterolemi
a
????????
?????? ---gt cell ?????????????? ---gt
???????????????
Insert DNA ?????????? infection by retroviral
gene therapy vector virus ????? normal LDL
receptor gene
Gene transfer
????????????????????? portal vein
LDL cholesterol ????
?????????????? 100 ???????? gene therapy
39
??????????????? gene therapy
????????
???????????
????????????????????????????? ????????????????gt
?????????? ??????????????????????????????????????
????????? ???????????????????????????????????????
?? ??????????????????????????????????????

• Germ-line gene therapy ??????????????????????????
• ????????????????????????????????????????????
• ??????????????????????? somatic gene therapy
  ????????? ????
• ??????????????????????????????????????????????????
  ??????

40
PREIMPLANTATION GENETIC DIAGNOSIS PGD
?????

• ????

?????????
????????????
???????????????????????? (PGD) ????????
??????????? ????????????????????????
(????????????????????)
(PRENATAL DIAGNOSIS) PND
41
PGD
??????????????????????????????????????? (PND) ???
????????????????????????? (AMNIOCENTESIS)
???????????????????????? (CHORIONIC VILLUS
SAMPLING)
???????????????????????? (CORDOCENTESIS)
42
PGD
?????????????????????????????
?????
?????
?????????????????????????
?????????????
???????????
???????????????????????????????????? (PGD)
43
PGD ????????????????? ??????????????????
???????????????? ????????????
???????????????????? ??????????????
?????????????
44
???????????????????? (IN VITRO FERTILIZATION IVF)
??????????????????????? ????????
?????????????????? (Female)
?????????????????? ??? ??????????????????
??????? ?????? (???????????)
?????????????????? (ESTRADIOL)
??????????????????????????????? 8-12 ??.
??????? 6-10 ?????
???????????????????? (???????????????)
????????? ????? ????????? 3 ???
45
PGD
???????????????????????????????????????
1) ???? FISH (Fluorescent In Situ Hybridization)
??????????????????????????????? --gt 13, 16, 18,
21, X, Y)
2) ???? PCR (Polymerase Chain Reaction)
?????????????? Single gene ( 50 ???)
46
?????????????????? Micromanipulator
47
???????????????????????? (biopsy)
???????/??????????????? (blastomere)
48
????????????????????? /??? ????????????
49
?????????????????????/???????????????
50
???????????????????? breast cancer
51
????????????????????????????breastcancer
52
????????? ---gt 3 ??????? ????????????????????
immune response
????????????????????????????????????
?????????????????????????????????????
53
???????????? e.g. Epstein Barr Virus (EBV),
Hepatitis B virus
?????????????????????????????????? ???????????????
?????
???????????????? virus ???? ---gt ???????????
?????????????????????????? ???????????????????????
?
54
CASE DISCUSSION
36 yr. ????????????????????? CA breast, ovary
???? BRCA 1 gene
???????????????????? ??????? 2 ???? ??? 1
?? ?????????????????
?????????? gene ?? mutate

55
????????????????????????? 45
36
???????????????? ??????????? 38, ??????????? 41
32
?????????? ??????????35