Transcript gene addition

GENETIC COUNSELING
AND GENE THERAPY
MODERATOR: Dr. Uday Kumar
Dr. Sahana Devadas
• “previously the geneticist was
like a ‘bookie’, offering odds for
any given event to happen. Now
that the geneticist is involved in
the ‘action’, i.e., diagnosis and
therapy, he has changed from a
bookie to a fixer.”
Roy D Schmickel
University of Michigan
Introduction:
• Genetic diseases are ubiquitous,
affecting all human beings where
ever they live. They place
considerable health and economic
burdens not only on affected people
and their families but also on the
community. As more environmental
diseases are successfully controlled,
those that are wholly or partly
genetically determined are
becoming more important
• Despite a general fall in perinatal
mortality rate, the incidence of
lethal malformations in newborn
infants remains constant.
• Between 2-5% of all liveborn infants
m have genetic disorders or
congenital malformations.
• Many common diseases in adult life
also have a considerable genetic
predisposition,including coronary
heart disease, diabetes, and cancer.
The prevalence of various
genetic diseases is given below:
Types of genetic
diseases
Estimated prevalence per
100 population
Single gene:
Autosomal dominant
Autosomal recessive
x-linked recessive
chromosomal abnormalities
common disorders with
2-10
2
1-2
6-7
appreciable genetic component
congenital malformations
7-10
20
Total
38-51
• In general human diseases may be
exclusively genetic or exclusively
environmental.
• Conditions like DMD and Down’s
syndrome are purely genetic in
origin. Whereas scurvy and TB are
purely environmental .
• Between the extremes many
common diseases like diabetes
mellitus , IHD, and congenital
malformations in which both genetic
and environmental factors are
involved and referred to as multi
factorial
• With emphasis on small family size ,
people are more concerned about
total well being of children and in a
situation like this, even rarer genetic
disorders should cause concern to
medical people.
• Further in south India where in
breeding has been practiced the
load of genetic disorders due to
autosomal recessive genes is high
and needs greater emphasis on
prevention and early treatment of
these disorders.
GENETIC COUNSELING
Definition
• It has been defined as an
educational process that seeks
to assist affected and / or at
risk individuals to understand
the nature of a genetic disorder,
its transmission, and the
options available to them in
management and family
planning.
Indications for genetic
counseling:
Advanced parental age:
• Maternal age > 35 years
• Paternal age > 50 years
• Child with congenital anomalies
or dysmorphology
• Consanguinity or incest
Family history of heritable disorders or
diseases , including:
• Adult onset
• Complex/multi factorial inheritance
• Chromosomal abnormality
• Single gene disorders
• Heterozygote screening based on
ethinicity, including:
• Sickle cell anemia (W.African,
Mediterranean, Arab,Indo- Pakistani,
Turkish , S.E Asian .
• Tay-sachs , canavan (Ashkenazi - Jewish ,
French - Canadian)
• Thalassemias (Mediterranean, Arab, IndoPakistani.)
Pregnancy screening abnormality,
including:
• Maternal serum alpha feto protein
• Maternal serum triple screen
• Prenatal ultra sound examination
• Still born with congenital anomalies
• Teratogen exposure or risk
Steps in genetic counseling:
• Diagnosis- based on accurate
family history, medical history,
• Examination and investigations
• Risk assessment
• Communication
• Discussion of options
• Long-term contact and support
Diagnosis:
• A full and accurate family history is
a corner stone in the genetic
assessment and counseling process.
• The 1st and most important step in
the diagnosis of genetic disorders is
construction of a family tree.
• The pattern of inheritance can be
shown from the pedigree . for eg:
vertical transmission in autosomal
dominant disorders, horizontal
transmission in autosomal recessive
disorders and oblique transmission
in X-linked recessive disorders
Autosomal recessive
X-linked recessive
Autosomal dominant
X-linked dominant
A
M
P
L
E
-Antenatal history
Maternal age,consanguineous
Marriage,Malformations,early onset
Malignancy
Previous child with genetic disorder,Proband
Loss of pregnancies
Exposure to teratogens, Early deaths
• All affected must be examined,
asymptomatic individuals should
also be examined to exclude mild or
early diseases.
• Home visits should be made, distant
relatives and older members of the
family should be interviewed and
special investigations like radiology,
cytology, DNA studies and histology
may have to be done.
Examination
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Anthropometry
Head to toe
Correct description of dysmorphology
Photographic record
Parental examination
Investigations
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Chromosome analysis
Biochemical analysis
DNA analysis
Histopathology
Team approach with the help from
other specialities like
orthopedicians , radiologists, and
ophthalmologists for arriving at a
proper diagnosis can be conducted.
Newer tests should be applied when
available for reconfirmation of
previous diagnosis.
RISK ESTIMATION
condition
Congenital heart
disease
diabetes
epilepsy
Severe mental
deficiency
Down syndrome
Cleft lip
Incidence/1000
Parents affected/not
3-8
Normal with 1 child affected
2
60
Normal with 1 child affected
5-10
One parent affected
5-15
5
30
1.5
1
One sibling
affected
Tri 1, 1 child affected T21/22 or
13-15/21 T21/21
1 child affected , 2 children
affected, 1 parent and child
affected
Manic depression
-
One parent affected
schizophrenia
8
0.06
-
One parent affected
Huntington chorea
Rh hemolytic disease
galactosemia
phenylketonuria
Recurrence risk
0.025
0.1
One parent affected
After 1 still birth, father
homozygous
One child affected
One child affected
5
1
33
3-5
10
17
15
9-20
50
100
25
25
• Recurrence risks should be
quantified, qualified and placed
in context
QUANTIFICATION
THE NUMERICAL VALUE OF A RISK
• The fact that the parents have just had a
child with autosomal recessive
disorder(recurrence risk equals 1 in 4)
does not mean that their next 3 children
will be unaffected.
• A couple faced with a probability of 1 in 25
that their next baby will have a neutral
tube defect should be reminded that there
are 24 chances out of 25 that their next
baby will not be affected.
Calculating and presenting the risk
• Hardy- Weinberg law
– By knowing the frequency of AR diseases ,
the frequency of carrier can be calculated
– P2+2pq+q2=1 where p is the frequency of
one of a pair of alleles and q is of others.
– Gives the frequency of carrier in the
population but not the recurrence risk.
Baye’s theorem
• Devised by Reverend Baye’s in 1763
• Provides the overall probability of an
event or outcome
• Provides assessment of recurrence risks
• Allows refinement of recurrence risk
estimates.
• Used in the interpretation of genetic
screening and diagnostic test results.
QUALIFICATION –THE
NATURE OF A RISK
• A ‘high’ risk of 1 in 2 for a trivial problem such as an
extra digit(polydactyly) will deter very few parents.
In contrast a ‘low’risk of 1 in 25 for a disabling
condition such as a neural tube defect can have a
very significant deterrent effect. A woman who
grew up watching her brother develop Duchenne
muscular dystrophy and subsequently die from the
condition aged 21 yrs, may not risk having children
even if there is only a 1% chance that she is a
carrier . other factors, such as whether it is
associated with pain and suffering, and whether
prenatal diagnosis is available , will all be relevant
to the decision –making process.
Placing risks in context
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For placing risks in context
1 in 10 is high risk,
1 in 20 as intermediate
And 1 in 50 as low risk
COMMUNICATION
• The ability to communicate is essential in genetic
counseling. It is a 2 way process. Both parents
should be present for the discussion , the genetic
basis for the problem should be described using
simple language and visual aids.
• Patients or the relatives should be encouraged to
clear their doubts on the condition.
• It is a good practice to record the communication and
send a letter summarizing the issues discussed.
• It should be non directive and non judgmental.
Directive counseling
• Directive counseling has a positive
influence on the consultees decision .
The non directive approach involves
presentation of the facts in an unbiased
manner leaving the entire responsibility
of decision with the consultee.
• Guilt should not be inculcated in a
parent as he or she has transmitted the
disease.
• Judgmental counseling may do opposite
effect as to abandoning the wife or
taking drastic decisions for divorce or
remarriage or not marrying
Discussion of options
LONG TIME CONTACT AND SUPPORT
Involvement of genetic counselor, family
practitioner,specialist genetic nurse,social worker and
enlistment of other medical specialities for
diagnosis,treatment,etc.. Should be ensured.
Support groups provide psychological support for families
by bringing them into in contact with the others with a
similar problem. All families with an affected member, and
carriers of specific genetic disorder should be put in touch
with relevant support groups where those exist. Such
groups identify the concerns of the families and allow the
community to participate in developing service. Long time
follow up should also be ensured via genetic registers. A
large number of community lay support groups have been
formed to provide information and to fund research on
specific genetic and non-genetic conditions.
Confidentiality
• Medical genetics team may learn many
family secrets , such as previous
abortions , previous abnormal births
and occasional false paternity.
• The team should observe high moral
values , confidentiality and should
respect the self esteem and moral
values of the parents.
OUTCOMES
• Most consultands have a reasonable
recall of the information given.
• 30% of the consultands have difficulty
in recalling the precise risk figure.
• 50%have been influenced by the
counseling in their reproductive
behavior.
Special problems
• Consanguinity and incest : consanguineous marriage
is one b/w blood relative who have at least one
common ancestor no more remote than a greatgreat-grandparent
• Union b/w 1st degree relatives (brother-sister)/
parent child is called incest.
• Most of the children born to consanguineous
marriage carries 2-6 lethal recessive mutations +1-2
AR mutations for harmful but viable traits.
Genetic
relationship of
partners
First degree
Proportion of
shared genes
Second degree
1/4
Third degree
1/8
1/2
Risk of
abnormality in
offspring
50%
5-10%
3-5%
Frequency of the 3 main types of abnormality
in children of incestuous relationship
Abnormality
Mental
retardation
Severe
Mild
AR Disorder
Congenital
malformation
frequency
25
35
10-15
10
ADOPTION & GENETIC DISORDERS:
 parents with high risk of genetic disorders like to adopt a
child
 counselors are called upon to determine the genetic
background of the child being placed for adoption.
MALFORMATION
Anencephaly/spina
bifida
cleft lip and palate
FREQ
/1000LB
4-5
Recurrence for
normal parents of
affected child
5
2
4-5
Cleft palate alone
0.5
2-6
Pyloric stenosis
2-3
3
CTEV
3-4
2-8
CDH
3-4
3-4
Hirchsprung disease
0.1
6
MOTHER OR FATHER WITH CHD:
Defect
AV septal
defect
AS
TOF
VSD
PS
ASD
Coarctation of
aorta
PDA
mother
14
Father
1
13-18
6-10
6
4-6.5
4-4.5
4
3
1.5
2
2
1.5
2
3.5-4
2.5
SIBLING WITH CHD
Defect
Recurrence risk
Endocardial fibroelastosis
4
VSD
3
PDA
3
ASD
2.5
TOF
2.5
PS
2
AS
2
Coarctation of aorta
2
ASD
2
Hypopalstic left heart
2
TGA
1.5
pulmonary atresia
1
Ebstein’s anomaly
1
Tricuspid Atresia
1
RISK OF RECURRENCE IN DOWN SYNDROME
Karyotype
father
mother Recurrence
risk(%)
translocation
21/22
N
C
10-15
C
N
C
N
N
N
C
N
N
N
5
100
100
1
small
21/21
Trisomy 21
Mosaic
Genetic centers in India
• 1. Center for genetic disorders , Department of Human
Genetics, Guru Nanak Dev university,Amritsar, Punjab
• 2. Dept of Human Genetics and Anatomy, St. Johns Medical
college Bangalore.
• 3. Dept of Pediatrics, K.E.M hospital. Mumbai.
• 4. ICMR Immunohematology center, K.E.M hospital. Mumbai.
• 5. ICMR Genetic research center, Wadia hospital for
children,Mumbai.
• 6. Dept of Genetics ,Ramakrishna Mission Hosp. ,Calcutta.
• 7. Depts of Pediatrics and Hematology PGI, Chandigarh.
• 8. Genetics unit, Dept of pediatrics, AIIMS, New Delhi.
• 9. Dept of Medical genetics, Sanjay gandhi postgraduate
institute of Medical science, Lucknow
• 10. Dept of Genetic Medicine, Sri Gangaram Hosp. Rajinder
Nagar, New Delhi.
• 11. Dept of Genetics, ICH ,Chennai
• 12. Genetic center, Dept of Pediatrics, BJMC, Pune.
Gene Therapy
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History
What is gene therapy?
How does it work?
Techniques of gene therapy
Candidate diseases
Factors for gene therapy to become effective
treatment for genetic disease.
Recent developments in gene therapy.
Current status of gene therapy
Arguments in favour of gene therapy
Arguments against gene therapy.
History of Gene therapy
• 1953: scientists Francis Crick & James Watsondetermined double helical structure of DNA.
• 1973: American doctor Stanfeild Rogers tried to
treat sisters with Hyperargininemia using human
pappiloma virus.
• 1980: Dr.Martin Cline first attempted at human
gene therapy in university of California,L.A.
• 1984:The human gene therapy working group
(HGTG) created.
• 1999:Death of Jesse Gelsinger , the first casualty
in gene therapy.
• 2002 August:successful treatment by
gene therapy of SCID “ bubble baby
syndrome”
• 2003 February: FDA’s biological
response modifier advisory committee
met to discuss possible measures to
allow retroviral gene therapy trials for
treatment of life threatening diseases.
TREATMENT OF GENETIC DISEASES
Environmental manipulation:
• Restriction
• Removal
• Replacement
Gene manipulation:
Gene therapy
Environmental manipulation
EXAMPLES OF METHODS FOR
TREATING GENETIC DISEASE
Treatment
Enzyme induction by
drugs .
Phenobarbitone
Replacement of deficient
enzyme/protein
Blood transfusion
BMT
Disorder
Congenital non –
hemolytic jaundice
SCID due to adenosine
deaminase deficiency
Mucopolysaccharidoses
Enzyme/protein
preparations
Trypsin
Trypsinogen deficiency
1-antitrypsin
1-antitrypsin
deficiency
Cryoprecipitate/Factor
VIII
-glucosidase
Hemophilia A
Gaucher disease
Replacement of
deficient
vitamin/coenzyme
B6
B12
Biotin
D
Homocystinuria
Methylmalonic
acidemia
Propionic acidemia
Vitamin-D resistant
rickets
Replacement of deficient
product
Cortisone
Congenital adrenal
hyperplasia
Thyroxine
Congenital
hypothyroidism
Dietary measures
Amino acids
Phenylalanine
Phenylketonuria
Leucine,isoleucine,valine Maple syrup urine d/s
Carbohydrate
Galactose
galactosemia
Protein
Urea cycle defects
Lipid
Cholesterol
Familial
hypercholesterolemia
Drug therapy
Aminocaproic acid
Angioneurotic edema
Dantrolene
Pancreatic enzymes
Malignant
hyperthermia
Familial hyper
cholesterolemia
Cystic fibrosis
penicillamine
Wilson d/s,cystinuria
cholestyramine
Drug/dietary avoidance
Sulphonamides
G6PD deficiency
barbiturates
Porphyria
Replacement of diseased
tissue
Bone marrow
transplantation
Removal of diseased
tissue
Colectomy
ADPKD, fabry disease
splenectomy
X-linked SCID, WiskottAldrich syndrome
Familial adenomatous
polyposis
Hereditary spherocytosis
What is gene therapy?
• Genes are the basic physical and functional
units of heredity.
• Genes are specific sequences of bases that
encode instructions on how to make proteins.
• It’s these proteins that perform most life
functions and even make up the majority of
cellular structures, not the genes
• When genes are altered so that the encoded
proteins are unable to carry out their normal
functions,genetic disorders can result.
• Gene therapy is a technique for correcting
defective genes,responsible for disease
development,where by the absent or faulty
gene is replaced by working gene so that the
body can make correct enzyme/protein and
consequently eliminate the root cause of
disease.
TECHNICAL ASPECTS
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Gene characterization
Target cells or organ identification.
Vector system
Incorporation of therapeutic gene into
host genome
• Production of desired protein
Approaches used for correcting faulty
genes:
• A normal gene, inserted into a non specific
location within the genome to replace a non
functional gene.
• Abnormal gene swapped for a normal gene
through homologous recombination
• Abnormal gene could be repaired through
selective reverse mutation which returns the
gene to its normal function.
• The regulation of a particular gene could be
altered.
How does gene therapy work?
• In most gene therapy studies-: a normal gene
is inserted into the genome to replace an
abnormal disease causing gene
• A carrier molecule called a vector must be
used to deliver the therapeutic gene to the
patient’s target cells
• Currently the most common vector is a virus,
that has been genetically altered to carry
normal human DNA.
• Viruses have evolved a way of encapsulating
and delivering their genes to human cells
• Scientists have tried to take advantage
of this capability and manipulate the
virus genome to remove disease
causing genes and insert therapeutic
genes.
TYPES OF GENE THERAPY
• Somatic cell gene therapy :
EX-VIVO
IN-VIVO
GERM LINE GENE THERAPY
Somatic cell gene therapy
germ line gene therapy
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 less ethical and social concerns
 not currently feasible due to moral, ethical and
scientific problems.
Could create a new unexpected human disease
or interfere with human evolution.
insertion of a vector(an agent
containing a modified gene)into a person’s
body to correct a genetic abnormality
it changes the genetic composition of
the person being Rxed and has no effect on
his/her future offspring as it only alters non
reproductive (somatic cell)
It usually targets one type of tissue within
the body e.g.: Ashanti Desilva case(SCID)
also called INHERITABLE GENE
MODIFICATION .
 Alters reproductive cells of a person’s body
corrects genetic defects in the person
treated, as well as in their future children.
 fertilized egg is used to inject plasmid
containing remedial gene(corrected or
replaceable gene)
Or remedial gene is inserted in retro viral
vector and transferred into fertilized egg.
Methods of somatic cell gene
therapy
• Invivo
• Exvivo
– Isolate cells with a
defective gene from an
affected individual
– Growing the isolated cells
in culture
– Correct the genetic defect
by transforming cells with
remedial gene
– Transplanting back these
cells into the patient. In
order to transfer the
remedial gene packaged
retro viral method is
employed
Example:
1.In adenosine deaminase (ADA)
deficiency.
2.In familial hypercholesterolemia
,
– Direct delivery of a remedial
gene into cells of a particular
tissue of an affected person
– Isolation of cells from patients
not required
– In gene construct, the
remedial gene represents a
sequence that codes a protein
that corrects the genetic
defect
– Remedial gene is under the
control of tissue specific
strong promoter. Some of the
viral vectors-(adeno, retro
virus) used to deliver
remedial gene inside the
patient
– Eg: cystic fibrosis,hemophilia
b
invivo
Cloned
gene
Gene transfer
exvivo
patient cells
Cultured cells
Return
to
patient
Select cells with
cloned gene
• Disease targets
Single gene defect
Severe combined
immunodeficiency
Gene(s involved)
Adenosine
deaminase
Tissues
Lymphoid tissue
-AT deficiency
-Antitrypsin
Lungs,liver(cirrho
sis)
Cystic fibrosis
CFTR
Lungs,pancreas
Hemophilia A & B
Factor VIII & IX
Blood clotting
Gaucher disease
Acid Macrophages,liver
glucosidase,gluco ,spleen,lungs
cerebrosidase
-globin
Hemoglobin
opathies
Blood formed
elements
Hyperchol LDL receptor
estremia
Liver,vascular
endothelial smooth
muscle cells
Phenylket Phenylalanine Liver
onuria
hydroxylase
Complex traits Genetic
approach
Cancer
Cytokine,HLA
genes.P53
HIV-1
RA
Tissue
Various
Antisense
Immune
constructs,imm system
unoenhancers
IL-1 recep
Synovial cells
.antagonist
Gene transfer to hematopoietic stem cells could be used to
treat diseases of erythroid, myeloid, and lymphoid cells as well as
platelet disorders (since the stem cell ultimately differentiates into all
these cell types)
In CHRONIC GRANULOMATOUS DISEASES , in which there is failure
of granulocytes and monocytes to generate the hydrogen peroxide
needed for bacterial killing, it may be feasible to achieve a threshold
level of normal cells to respond to infections.
Another major area of stem cell gene therapy research is the treatment
of HAEMOGLOBINOPATHIES with vectors expressing globin genes
Lymphocyte gene transfer has the potential to treat GENETIC
IMMUNODEF ICIENCIES and to modulate immune functions. ADA
deficiency was one of the first diseases to be treated with gene therapy
and employed ex vivo transduction of lymphocytes
Genetic manipulations with antibody or cytokine genes offer a multitude
of possible treatments for infectious and autoimmune diseases, as well
as for CANCER.
COAGULOPATHIES
Inherited coagulopathies, especially HAEMOPHILIA A&B ,
Hemophilia A has been more difficult to treat, due to the
larger cDNA (which approaches the packaging capacity of
AAV vectors) and the requirement that expressing cells
deliver the protein directly into the intravascular space.
Other coagulopathies, such as FACTOR X DEFICIENCY
currently in the early stages of clinical trials.
LIVER AND GASTROINTESTINAL TRACT
•Many genetic diseases amenable to gene replacement in hepatocytes. Due
to regenerative capacity of the hepatocyte, integration of a vector offers the
possibility of lifelong gene expression.
•The first hepatic gene therapy attempted for -treatment for familial
hypercholesterolemia.
•Adenovirus vectors are very effective at gene transfer into the liver, thus
allowing for the transduction of a majority of hepatocytes.
•AAV vectors stably integrate their proviral DNA into hepatocytes in vivo
with no apparent toxicity. -useful for treating a variety of metabolic diseases,
such as urea cycle disorders, aminoacidopathies, disorders of carbohydrate
metabolism, and lysosomal storage diseases.
• The cardiovascular system
(including the peripheral
vasculature) has become an
important target for gene
therapy.
•
to inhibit smooth-muscle cell
proliferation and PREVENT
RESTENOSIS.
•
to promote the vascularization
of tissues by intramuscular
injection of naked DNA vectors
encoding the vascular
endothelial growth factor
(VEGF) gene in patients with
with critical LIMB ISCHAEMIA
due to poor peripheral
vascularization
PULMONARY SYSTEM
Gene therapy of the lung has concentrated primarily on
treatments for cystic fibrosis.
The gene for cystic fibrosis, CFTR, was cloned in 1989; by
1993 the first trials using adenovirus vectors were attempted
in the nasal epithelium and airway.
clinical trials with nonviral liposome and AAV vectors failed
to demonstrate therapeutic effects.
Difficulties encountered * include poor vector delivery due to respiratory
secretions,
* lack of accessible vector receptors on the
exposed cellular surface,
* transient transgene expression due to
turnover of the respiratory epithelium,
* vector-induced inflammation and pneumonitis.
NERVOUS SYSTEM AND RETINA
Gene transfer to the nervous system will be important for the
treatment of many inherited and acquired neurologic diseases.
Depending on the disorder, both glial cells and neurons may be
appropriate cellular targets
•PARKINSONS DISEASE, where transduction in the striatum could
allow selective expression of genes involved in dopamine
synthesis.
•The possibility of delivering neurotrophic factors to the CNS is a
potential strategy for the treatment of neurodegenerative diseases,
such as AMYOTROPHIC LATERAL SCLEROSIS,
• for facilitating recovery after SPINAL CORD INJURY.
•RETINITIS PIGMANTOSA
CANCER
. One approach uses gene therapy with
cytokine or neoantigen genes to
INCREASE TUMOR
IMMUNOGENICITY. The vector is
usually injected directly into the
tumor, and there is some evidence
that once the immune system is
stimulated, nontransduced tumor
cells may also be eliminated by the
immune system.
Genes that CONTROL TUMOR GROWTH
when expressed in nontumor cells
may also be effective in cancer gene
therapy.
interfere with tumor ANGIOGENESIS.
. Finally, lytic viral vectors that
selectively replicate and kill
malignant cells are being developed.
One example is an adenovirus
designed to replicate in cells deficient
in p53, a tumor-suppressor protein
that is mutated in many different
cancers
Vectors used for gene therapy
• Viral options for gene delivery
– Retrovirus
– Adenovirus
– Adeno associated virus
– Herpes simplex virus
– Vaccinia
– Influenza
Vectors used for gene therapy
• Non viral options
– Plasmid DNA
• Naked
• Liposome
• Ligand-DNA complex
Non targeted
Targeted
– Transkaryotypic therapy
– Calcium phosphate precipititation
Ideal vector
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Capable of direct in vivo administration
Targeted delivery to specific cell
Safely integrated into genome
Transferred to all daughter cells
Site of insertion should be specific and should
include excision of defective gene and its
replacement by normal gene
• Integrated into non oncogenic sites
• Infection should not cause cell lysis.
• Currently no vector satisfies most of
these criteria
DNA clone
Package in virus
Transfect into host cell
DNA integrates into
a chromosome
Replication and inheritance of
DNA by daughter cells, Long term
expression
Protein made
DNA remains extra
chromosomal in nucleus
Short term
expression
Protein made
Gene Delivery Strategies for Gene Therapy
LONG TERM
TRANSIENT
* hemophilia
* killing cancer cells
* sickle cell anaemia
* preventing
coronary restenosis
* providing DNA
based immunisation
* impairing viral
replication
Vectors used for gene therapy
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Viral options for gene therapy:
Retroviruses
Adenoviruses
Adeno associated viruses
Herpes simplex viruses
Retro virus
• RNA virus with reverse transcriptase
• Moloneymurin leukemic virus & Gibbon leukemic virus
are most widely used.
benefits:
100% transduction
Can infect variety of cell lines
Does not lead to cell lysis
Precise integration * cellular DNA is possible
Long term expression – integration* chromosomal DNA
limitations
• Cell receptors are required and most
retro viral recepters are not identified.
• Requires cell division
• Potential for insertional mutagenesis
• Limited size of DNA insert
• Potential recombination of therapeutic
virus with endogenous retro viruses
that can be pathogeneic
Adenovirus
• Large
double stranded DNA virus
• Natural viral pathogen to human being
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Benefits:
Infects non dividing cells
Large segments of DNA can be transported
Low risk of insertional mutagenesis
Broad range of target cells.
Efficient in –vivo delivery
Low risk of oncogenesis
Limitations
• Can lead to cell lysis
• Doesn’t stably integrate into the
chromosome, but remains as episomes
• Transient expression. Gradually lost
• Immunogenic – major limiting factor
• Can initiate inflammatory response
Adeno associated virus
• Small DNA containing parvovirus
• Requires adeno virus for replication(coinfection)
• Replicates as double stranded DNA but
packed as single stranded DNA
• Integrates into specific location on human
chromosome 19 , which is linked to B- cell
CLL
• Less efficient & less precise
• Does not require cell division
Others
• Herpes virus: large double stranded
DNA virus
• Exists as episomes in the target cells.
• Can accommodate a large gene
• Useful for the introduction of genes in
CNS
• Vaccinia and influenza are in
experimental stage.
Non- viral options
• Direct injection of naked DNA
• Plasmids are incorporated into liposomes( synthetic
cationic lipid)
• No specific receptors needed.
• Ligand DNA complex:targeted gene delivery.
• Plasmid DNA and specific polypeptide ligand complex
are generated
• Taken up by the process of endocytosis by cells.
• Incorporated into the DNA
• Limiting factor is escape of DNA from endosomes to
nucleus.
Trans karyotypic therapy
• A small sample of patients’ cells are
removed ,
• genetically modified with the gene of
therapeutic by a process called electro
poration.(using a brief electrical pulse in
open pores in the cells)
CaPo4 precipitation
• Enters cell by endocytosis and
incorporated into the nucleus.
• Advantages:
• No infection risk
• Completely synthetic
• No limitation of insert size.
Disadvantages:
• Low efficacy
• Limited target cell range
• Transcient expression
Candidate diseases for gene therapy
• Gene therapy is likely to have the greatest
success with diseases that are caused by
single gene defect
• By the end of 1993, gene therapy had been
approved for the use on diseases like:
 Severe combined immunodeficiency
 Familial hypercholesterolemia
 Cystic fibrosis
 Gaucher’s disease
• Most protocols to date are aimed
towards the treatment of:
Cancer
Aids
Parkinson’s disease
 Alzheimer's disease
Arthritis
Heart disease
• The human genome project an ongoing effort
to identify the location of all genes in the
human genome continues to identify genetic
diseases.
• The USA government will provide $200 million
each year to scientist in multi disciplinary
research center who are attempting to
determine/identify the make up of all human
genes
• Similar programme is also there in Europe.
Criteria for selection of disease candidate for human
gene therapy- eve nicholas
• The disease is incurable, life threatening
• Organ, tissue & cell types affected by the
disease have been identified
• The normal counter part of the defective
gene has been isolated & cloned
• Normal gene can be introduced into a
substantial sub- fraction of the cells from the
affected tissue or that introduction of the
gene into the available target tissue, such as
bone marrow, will some how alter the disease
process in the tissue affected by the disease.
• The gene can be expressed adequately.
• Techniques are available to verify the
safety of procedures
Some protein products of
recombinant DNA technology
product
Made in
use
Human insulin
E-coli
Treatment for
diabetes
Human growth
hormone(GH)
E-coli
Treatment for
growth defects
Epidermal growth
factor (EGF)
E-coli
Treatment for burns,
ulcers
cellulase
E-coli
Breaking down
cellulose for animal
feeds
taxol
E-coli
Treatment for
ovarian cancer
Interleukinn-2(IL-2)
E-coli
Possible treatment for
cancer
Bovine growth
hormone(bgh)
E-coli
Improving weight
gain in cattle
Interferons
(alpha&gamma)
s.cerevisiae;E-coli
Possible treatment for
cancer & viral
infections
Hep-B vaccine
s.cerevisiae
Prevention of viral
hepatitis
Erythropoietin(EPO)
Mammalian cells
Treatment for anemia
Tissue plasminogen
activator(TPA)
Mammalian cells
Treatment for
hemophilia
Factor VIII
Mammalian cells
Treatment for heart
attacks.
Factors which have kept gene
therapy from becoming an effective
treatment
•
•
•
•
Short-lived nature of gene therapy
Immune response
Problems with viral vectors
Multi-gene disorders
RECENT DEVELOPMENT IN GENE THERAPY
• University of California, Losangeles,research
team gets genes in to the brain using
liposomes coated in a polymer –polyethylene
glycol(PEG).
• Transfer of genes in to the brain is a
significant achievement because viral vectors
are too big to get across the “blood brain
barrier” .This method has potential for
treating Parkinsons disease .
• RNA interference or genes silencing
may be a new way to treat
Huntingtons disease.
• New gene therapy approach ,repairs
errors in m-RNA derived from defective
genes.
• Techniques has potential to treat
Thalassemia ,Cystic fibrosis & some
cancers.
• Gene therapy for treating children with XSCID or “BUBBLE BOY “ disease is stopped in
France, when the treatment caused leukemia
in one the patients.
• Researcher`s at Western Reserve University
& Copernicus Therapeutics are able to create
Tiny Liposomes 15nm`s across that can carry
therapeutic DNA through pores in the nuclear
membrane.
• Sickle cell is successfully treated in mice.
CURRENT STATUS OF GENE THERAPY
• FDA has not yet approved any human
gene therapy product for sale.
• Current gene therapy is experimental
and has not proved very successful in
clinical trials.
• In 1999, gene therapy suffered a major
set back with a death of 18yr old
JESSE GELSINGER (OTC deficiency)
• Another major blow came in 2003, when
the FDA placed a temporary halt on all
gene therapy trails using retro viral
vectors in blood stem cells
• FDA took the action after it learnt that a
2nd child treated in French gene therapy
trail had developed leukemia like
condition.
•
Researchers also are experimenting with introducing a
47th (artificial human) chromosome into target cells.
This chromosome would exist autonomously
alongside the standard 46 --not affecting their
workings or causing any mutations. It would be a
large vector capable of carrying substantial amounts
of genetic code, and scientists anticipate that,
because of its construction and autonomy, the body's
immune systems would not attack it. A problem with
this potential method is the difficulty in delivering
such a large molecule to the nucleus of a target cell.
Arguments in favor of gene therapy
• Can be used to treat desperately ill patients
or to prevent the on set of horrible illness.
• Conventional treatment has failed for the
candidate diseases for gene therapy & for
these patients gene therapy is the only hope
for future
• Eric Juengst summarized the Arguments in
favor of and against human germ line gene
therapy.
• Germ line gene therapy offers a true cure &
not simply palliative or symptomatic
treatment
• Germ line therapy may be the only
effective way of addressing some
genetic diseases .
Arguments against the development
of germ line gene therapy.
• Germ line gene therapy experiments would
involve too much scientific uncertainty &
clinical risks & the long term effects of such
therapy are unknown.
• As germ line gene therapy involves research
on early embryos and affects their offspring.
Such research essentially creates generations
of unconsenting research subjects.
• Gene therapy is very expensive and will never
be cost effective enough to merit high social
priority.
• Germ line gene therapy would violate
the rights of subsequent generations to
inherit a genetic endowment that has
not been intentionally modified.
Some questions to ponder
•
•
•
•
•
When should gene therapy be used? Should it be used to treat
critically ill patients? Should it be used to treat babies and children?
What effect would gene therapy have on future generations if
germline (reproductive) cells were genetically altered? How
might this alteration affect human variation?
Who should decide what are "good" or "bad" uses of genetic
modifications? How do you define "normal" with regard to human
beings?
What if we could alter human traits not associated with
disease? Would it be okay to use gene therapy to improve or enhance
a person's genetic profile?
Who will have access to gene therapy, treatments and longterm follow-ups? Will gene therapy and genetic enhancements
create an advantage for those who can afford it?
• The questions raised here have no clear right or
wrong answer. Your responses will depend on your
values, as well as on the opinions of those around
you.
References
• Elements of medical genetics;10th edn,Mueller
&Young.
• Essentials of medical genetics; 4th edn, Connor&
Ferguson smith.
• Principles of medical genetics;2nd edn.
Gleehrter,Collins& Ginsburg
• Genetics counseling in pediatric practice: Phadke
&Phadke,Ind. Ped.
• Memorix pediatrics; Dieter Harms & Jochem Scharf
• Nelson textbook of pediatrics 16 th edn
• Harrison’s principles of internal medicine 15th edn
• Textbook of pediatrics Forfar 5th edn
• Genetic disorder by M L Kulkarni.
GENE
ADDITION
Cystic fibrosis
Familial hypercholesterolemia
Hemophilias A and B
Thalassemia
Immunodeficiencies
Metabolic disorder
Duchenne's muscular dystroph
Retinitis pigmentosa
Express CFTR in pulmonary system
and/or GI tract
Express low-density lipoprotein
receptor in liver
Express factor VIII or IX and secrete
in circulation
Express normal globin in red blood
cells
Express mutant genes, such as
adenosine deaminase
Express missing enzymes or
transporters
Express mutant dystrophin protein in
muscle cell
Express normal protein in retina
Gene correction
Lesch-Nyhan
Retinitis pigmentosa (dominant)
Sickle cell disease
Cystic fibrosis
Modify hypoxanthine
phosphoribosyl transferase
locus
Correct missense mutation
Correct -globin mutation
Correct DF508 mutation in
pulmonary system
Modify vascular biology
Cardiovascular diseases
Coronary artery
restenosis
Peripheral vascular
disease
Hypertension
Block cell
proliferation in
vessel wall
Induce angiogenesis
Express genes (e.g., tissue
kallikrein) to induce
vasodilation
THANK YOU