Transcript Hemophilia case Stud - Lectures For UG-5

Group 3
Fabiha Aziz
Fariha Munir
Fatima Tuz Zahra
Fatima
Tuz
Khan
Muhammad Ahmad
Saneea Imran
Contents
1.Introduction of the disease or disorder
2.Types of mutations involved
3.Conventional treatment
4.Type of vector
5.The procedure of gene therapy
6.History of clinical trials
7.Commercially available treatments
Introduction
• What is hemophilia?
• What is does to our body?
• What causes hemophilia?
What is hemophilia?
• Hemophilia is an inherited bleeding disorder
in which the blood fails to clot
effectively.
• Hemophiliacs do not bleed more extensively
or more quickly than the normal people but
they do bleed for a longer time after an
injury or surgery.
What it does to our body?
• The bleeding may be internal or external
• Internal bleeding occurs in knees ankles
tissues and muscles. If left untreated the
bleeding in the joints may lead to agonizing
pain
• And can be fatal when the bleeding occurs in
the vital organ like brain
Types of Hemophilia
Hemophilia C is
Hemophilia B is
an autosomal
Hemophilia A is a recessive Xgenetic
a recessive Xlinked genetic
disorder
disorder
linked genetic
involving lack
disorder
involving a
of functional
lack of
involving a
Clotting Factor
lack of
functional
XI.
clotting Factor
functional
clotting Factor
IX. It
Hemophilia C is
comprises
VIII and
not completely
represents 80%
approximately
recessive, as
of hemophilia
20% of
heterozygous in
hemophilia
cases.
dividuals also
cases.
show increased
bleeding.
Hemophilia A
• All races
• Affects men and women become carriers as
Severe
geneInis present
on the X chromosome.
cases
of
hemophilia
the blood has
less than 1%
of
the
clotting
protein
hemorrhages
occurs
several
months
no
apparent
cause
Moderate
cases minor
traumas are
usually the
cause of the
cause of
bleeding
Milder have
fewer
hemorrhages
Factor Replacement Therapy
Infusion of factor
8-9 to prevent or
control bleeding
Plasma derived
factors and
genetically
engineered
MUTATION
By: Fatima Tuz Zahra
Mutation of Hemophilia A
• Coagulation factors are proteins that work
together in the blood clotting process.
• F8 gene provides instructions for making a
coagulation factor VIII
• Mutations in the F8 gene lead to the
production of an abnormal version of
coagulation factor VIII.
28 region of X Chromosome.
Inversion and Crossing Over of Intron22
Conventional Treatments
By: Tatheer Fatima
Conventional treatment
The two main approaches to treatment are
• Preventive treatment :
Refers to the regular medications that are
taken to prevent episodes of bleeding and
subsequent complications
• Episodic treatment :
Used to manage episodes of prolonged bleeding.
Conventional treatment
• Treatment With Replacement Therapy :
Concentrates of clotting factor VIII
(hemophilia A) or clotting factor IX
(hemophilia B) are slowly dripped or injected
into a vein
• Preparation of clotting factor concentrate :
Human Blood Clotting factor concentrate
 Recombinant Clotting factors
Conventional treatment
Types of Replacement therapy :
• Preventive or prophylactic therapy:
Replacement therapy on a regular basis to
prevent
bleeding
• Demand therapy:
only need replacement therapy to stop bleeding
when it occurs
Conventional PROS
treatment
1. Quicker treatment.
Early treatment
lowers the risk of
complications
2. Fewer visits to the
doctor or emergency
room
3. Costs less than
treatment in a
medical care
setting
4. Helps children
accept treatment
and take
responsibility for
CONS
1. Antibody(proteins)
that attack the
clotting factor
2. Viral infections
from human
clotting factors
3. Damage to joints,
muscles, or other
parts of the body
resulting from
Conventional treatment
Desmopressin
• Man made hormone
• Can treat mild hemophilia A
• Cannot treat hemophilia B or severe hemophilia
A
• Stimulates the release of stored factor VIII
• Injection or as nasal spray
• Only in certain situations. e.g. you may take
this medicine prior to dental work or before
playing certain sports to prevent or reduce
bleeding
Conventional treatment
Anti fibrinolytic Medicines
• Tranexamic acid and epsilon aminocaproic acid
• Can be used with replacement therapy
• Given as a pill and they help keep blood clots
from breaking down.
• Most often are used before dental work or to
treat bleeding from the mouth or nose or mild
intestinal bleeding.
Types of vector
By: Muhammad Ahmed
Ex-vivo
• Cells transduced in culture, then returned
to patient
• Greater control over transfection conditions
• Unchanged cells can be screened out
• Difficult to transpant
In-vivo
• Cells modified within the body
• Introduction of vector with genetic material
in patient’s body
• No transplantation issues
• Cost-effective
• May provoke immune system
• Ultimate goal for gene therapy
Viral Vectors
Retroviral vectors
• Moloney murine leukemia virus (MoMLV)-based
• Can infect wide variety, integrate into host
genome, relatively non-immunogenic
• Only in actively dividing cells
• Problems: insertional mutagenesis,
inactivation by complement
Adenoviral vectors
• Relatively large, double-stranded DNA
viruses
• Infect non-dividing cells
• Can transfer multiple copies of gene, in
vivo
• Problems: Many humans may be immune to
virus, not integrated into host genome
Adeno-associated (AAV) vectors
• Relatively small, single stranded DNA
parvovirus
• Viral coding sequence replaced by transgene
• Non-dividing cells
Lentiviral Vectors
• Integrate into genome, infect non-dividing
cells
• Problems: limited range of cell targets,
pathogens!
• HIV – big risk of recombination
infectious virus
Non-Viral Vectors
• Through plasmids
• Problem: Transient expression of genes
(several days)
• Direct injection of naked DNA
• Problems: Low efficiency of transduction,
not integrated into genome
Procedure of Gene Therapy for
Hemophilia A
By: Saneea Imran
Gene therapy for hemophilia A
• Hemophilia is an interesting target for gene
therapy as the genetics of the disease are
well understood.
• Introducing a functional copy of the mutated
gene through a vector can provide an
effective cure.
Clotting factor level =
Clinical Phenotype
Gene therapy for Hemophilia A
• Vector: Both viral and non-viral vectors can
be used
• Target Cell: Hematopoietic stem cells,
hepatocytes, skeletal muscle cells,
endothelial cells.
• Approach: Both in-vivo and ex-vivo gene
therapy approach can be used.
Non-Viral vector for gene therapy of Hemophilia
A
• Year: 2001
• Non-viral somatic cell gene therapy for
hemophilia A
• Vector used: Plasmid
• Cells targeted: Dermal fibroblasts
Procedure
Desired gene that
produces Factor VIII
Restriction
enzymes
Preparing the vector
Gene
of
interest
Cells that
successfully
produce FVIII
are selected
and inserted
into patient
Transfection
Antibiotic
gene
Dermal fibroblasts of
patient
( skin biopsy )
Patient with
Hemophilia
Viral Vectors for gene therapy of Hemophilia A
Vectors that have been used are:
1. Retroviral and Lentiviral Vectors
2. Adeno Associated Viral Vectors
Using Retroviral Vectors
Producing recombinant Retroviral vectors
Targeting cells with Retrovirus
Clinical Trials
By: Fatima Tuz Zahra Khan
Stem cell-based gene transfer
• Implantation of Genetically Modified Stem
Cells
• Genetic modification and transplantation of
hemopoietic stem cells (HSCs)
• Use of Retroviral and lentiviral vectors
• Trials remain un-successful due to
insufficient levels of FVIII
In Vivo methods
Retroviral vectors
• Retroviral vectors transduce replicating cells
by stably integrating into the host genome.
• Preclinical studies involving rabbits and dogs
did show possible benefits.
Routes?
• Subjects received a peripheral intravenous
infusion of retroviral vector carrying a B
domain–deleted human FVIII gene.
Efficiency?
• 9 of 12 subjects showed a FVIII level above 1%
• Insertional mutagenesis and the risk of
LENTIVIRAL VECTORS
• Lentiviruses efficiently transduce
nondividing cells.
• Overcoming a hurdle faced by retroviral
vector systems.
• Routes?
• In murine models, intraperitoneal
administration of lentivirus transduced
liver, spleen, blood and bone marrow
• Efficieny?
• four- to sixfold rise in expression
ADENOVIRAL VECTORS
• One of the most efficient vectors
• Transducing dividing and nondividing cells, with
a tropism for hepatocytes
• Genetic payload capacity making them ideal
vectors for the FVIII gene
Routes?
• Intramuscular Injection.
• Liver-directed administration
Efficiency?
• Single Human Trail
Adeno associated Virus:
• Success in mouse and other large animal models.
• Limitations due to small genome size.
Routes?
• Intramuscular Injection.
• Liver-directed administration
Efficiency?
• Two trials have examined the use of AAV-2
vectors in human subjects
• Limited efficacy
Commercially available
Treatments
Treatment
• Regular infusions of DDAVP or clotting factor
• Clot-preserving medications (antifibrinolytics)
• Fibrin sealants
• Physical therapy
• First aid for minor cuts
• Vaccinations
Clinical Trials
• Currently are for hemophilia B
• AAV vector encoding factor IX (FIX) under
the control of a liver-restricted promoter
• Promising results
Problems
• First clinical trial of hepatic artery
infusion of an AAV2-FIX vector quickly
encountered obstacles that had not been
identified in the preclinical studies
• Risk of vertical transmission
• Risk for potential offspring if the
spermatocytes are transduced
References
• Haemophilia A: from mutation analysis to new therapies
• Jochen Graw, Hans-Hermann Brackmann, Johannes Oldenburg, Reinhard
Schneppenheim, Michael Spannagl & Rainer Schwaab
• Genetics Home Reference
• (http://ghr.nlm.nih.gov/condition/hemophilia)
• http://www.visibleproductions.com/index.php?page=asset_detail&asset_id=vpl_
0715_001