Transcript Document

‫محاضرات‬
‫األستاذ الدكتور‬
‫محمد عبد اللطيف‬
Gene
Therapy
Presented by
Professor
Mohamed Abd Ellatif
Professor of Medical Biochemistry
And Molecular Biology
Faculty of Medicine, Mansoura University
Mansoura, Egypt
Definition of Gene therapy
Gene therapy is an
experimental technique that
uses genes to treat or prevent
disease.
Approaches of gene
therapy
 Replacing a mutated gene that causes
disease with a healthy copy of the gene
 Inactivating, or “knocking out” a mutated
gene that is functioning improperly.
 Introducing a new gene into the body to
help fight a disease.
Principle
 Introduction
of genetic material
into cells to compensate for
abnormal genes or to make a
beneficial protein.
Two approaches for delivering
genetic material exist
• In vivo gene therapy
• Ex vivo gene therapy
In vivo gene therapy

Direct delivery of DNA (usually via a viral
vector) to resident cells of the target tissue.

There are two requirements for such a strategy:
1. The target cells is easily accessible for infusion or
injection of virus.
2. The transfer vector infects, integrates, and then
expresses the therapeutic gene in target cells (not
surrounding cells) at effective levels for extended
time periods.
Approach of in vivo
gene therapy
The vector can be injected or
given intravenously directly into
a specific tissue in the body,
where it is taken up by individual
cells.
Vectors used in gene therapy
• Viruses
• Non-viruses
Viruses used are:
Retroviruses
Adenoviruses
Adeno-associated viruses
Lentivirus
Disadvantages of viruses as
vector in gene therapy

In all viral types, the vectors tend not to
disperse well in a targeted tissue. Even
when injected directly into a tumor, they
are prone to miss some of the targeted
cells.

In addition, their use does not allow longterm gene expression.
Non-viral vectors
• Direct introduction of therapeutic DNA
into target cells.
• Creation of an artificial lipid sphere
with an aqueous core (liposome).
• Chemically linking the DNA to a
molecule that will bind to special cell
receptors.
• Human artificial chromosomes (HACs)
Liposome
Definition

are artificial vesicles with a phospholipid
bilayer membrane.

It is self-closing spherical particles where
one or several lipid membranes
encapsulate part of the solvent in which
they freely float in their interior.
Size

liposomes are typically 5-10 µm in
diameter with the phosopholipid bilayer
about 3 nm thick
Mechanism of
formation

self-assembly process that is driven
by the amphipathic nature of
phospholipid molecules
Function
 A liposome can be used to deliver
drugs, proteins or nucleic acids (short
stretches of DNA and plasmids
encoding therapeutic genes) to a cell.
Mechanism of action of
liposome
First the outer layer of the liposome fuses
with the outer layer of the plasma
membrane.
 Second, the two fused membranes
coalesce as the inner layer of the liposome
approaches the inner layer of the plasma
membrane.
 Finally, the two inner layers fuse so that
the drug has access to the cytoplasm.

• Methods for Enhancing The
Efficiency of
Liposome-Based Transfection
The efficiency of lipid-mediated
gene transfection is dependent on
several steps, including:

Adsorption of the transfection complex to
the cellular surface

Escape from the endosome/lysosome

Translocation across the nuclear
membrane and into the cell nucleus where
transcription occurs
To improve translocation across
the nuclear membrane
nuclear localization signals (NLS) are
used:
• classical NLS
• non-classical M9 NLS
Artificial chromosomes
Advantages of adding an
entirely separate artificial
chromosome

Eliminates the risk of DNA landing in a
wrong place on cell's genome, which can
trigger cancer.

The ability to deliver multiple therapeutic
genes, but viruses can carry only short
sequences of DNA.
 This artificial chromosome behaves like
a normal one in mice: it is duplicated
when cells divide and is passed from
generation to generation.
 The human artificial chromosome
survived for as long as 6 months in
cells, retaining its integrity while
replicating during many cell divisions.
Structure

Capping the ends of chromosomes are
telomeres, which is brief repeating
sequences of DNA.

Origins of replication, which is DNA
sequences that initiate the replication of
a chromosome during cell division.
 At the center of each
chromosome is the mysterious
centromere, which plays a vital
role in the chromosome's
segregation in a dividing cell.
 Ignorance of the structure, and
the size, of human centromeres
has been the main reason of
inability to create human artificial
chromosomes.
Disadvantages of in
vivo gene therapy:
 The
introduced gene will
integrate in the genome and
cause interruption or disruption
of other gene functions, causing
mutation and possibly disease.
Ex vivo gene
therapy
Approach
Ex vivo gene
transfer. The ex
vivo strategy is
based on the
utilisation of a
surrogate cell that
is infected with
virus in vitro.
The surrogate cell
is subsequently
transferred to the
target tissue and
expresses the
therapeutic gene .
For successful ex vivo, The cells
used should be:
•
•
•
•
Readily available and relatively easily
obtained.
Able to survive for long periods of time in
vivo.
Able to express a transgene at high
levels for extended durations.
Not elicit a host mediated immune
reaction.
The advantages of using an
ex vivo approach
Selection of the modified cell population
before transplantation.
Subclone cells and produce monoclonal
populations that produce high levels of
therapeutic protein.
• The ability to screen populations and
exclude the presence of helper viruses,
transformational events, or other
deleterious properties obtained after
or during the modification process.
• So, Viral vectors of low transfection
efficiency can be used, because
uninfected cells can be selected out of
the transplant population.
Currently, autologous
primary cell cultures
are used.
Autologous means that
the donor and
recipient organism
are the same
The advantage of using primary,
autologous cell cultures include:
• Lack of antigenicity.
• Decreased risk of malignant
transformation.
Disadvantages of using
primary, autologous cell
cultures include

Difficulty in harvesting some types of
primary cells, maintaining them in culture,
and effectively expressing transgenes
through current transfection techniques.
 Another
complication arises when
primary cells are transferred to
non-host tissue.
 for example, primary fibroblasts
transplanted to the CNS will often
produce collagen and other skin
appropriate products that interfere
with normal CNS functioning.
 This
problem may be overcome
with the use of stem cells.
Stem cell ex vivo gene
therapy:
• Bone marrow derived stem
cell.
• Hepatocytes.
• CNS stem cells
• Fetal derived stem cells.
Human embryo at 4 cell
stage
Stem cells used in treatment of
neuro-degenerative diseases
(Infarction)
Heamobiotic Stem cell
Fetal Stem Cells
Blastocyte
Approach Stem cell ex vivo gene
therapy

Peripheral derived haematopoietic stem cells
are of particular interest as a potential
surrogate cell.

Haemopoietic stem cells are easily obtained
through I.V route, harvested systemically,
modified in vitro, re-infused into the
peripheral blood with subsequent homing to
damaged target tissue such as brain or
myocardium.
Advantages
 Adult stem cells completely incorporate into
any host tissue and transform into a mature
cell of that organ.
 This ability ensures long term survival of
grafted cells.
 So these cells could be used to carry
therapeutic proteins, and also to
repopulate organs with damaged or
depleted cell numbers.
Disadvantages

Low viral transfection efficiency.

Technical difficulties in isolating,
culturing, and maintaining these
cells.
• How safety is the gene
therapy

Gene therapy have very serious health risks,
such as toxicity, inflammation, and cancer
which is due to the administration of
retrovirus, which incorporates randomly into
the genome and can lead to insertional
mutagenesis and malignant transformation.
Factors have kept gene therapy
from becoming an effective
treatment for genetic disease
•
Short-lived nature of gene therapy
•
Immune response
•
Problems with viral vectors as toxicity,
immune responses, gene control,
targeting tissues and reactivation of
virus.
• Lack of viral specificity
• Multigene disorders as Alzheimer’s
disease, DM and heart disease.
• Inefficient gene transfer
Inability to control gene
expression:
 Induction of inflammation for treating such
diseases as cancer may be useful, but once
the cancer is cured the inflammation
continues if cells are expressing the inciting
transgene. Chronic inflammation of a
specific tissue is undesirable.
The use of growth factors
Uncontrolled growth factor expression
and function is intimately involved in
the malignant transformation
processes.
 The contineous expression of a growth
factor predisposes to malignancy.
 It is essential to be able to turn off
growth factor expression if malignancy
is detected, or if treatment is toxic or
no longer useful or necessary.

Recent developments in gene
therapy research
• Injection of genes into the brain using
liposomes coated in a polymer call
polyethylene glycol (PEG). Viral vectors
are too big to cross the blood brain
barrier. This is important in treating
Parkinson’s disease
• RNA interference or gene silencing may
be a new way to treat Huntington's.
•
New gene therapy approach repairs
errors in messenger RNA derived from
defective genes.
•
Gene therapy for treating children with XSCID (sever combined
immunodeficiency) or the "bubble boy"
disease is stopped when the treatment
causes leukemia in one of the patients.
•
Creation of tiny liposomes 25
nanometers (the already Known is 510 µm) across that can carry
therapeutic DNA through pores in the
nuclear membrane.
•
Sickle cell is successfully treated in
mice.
Approaches of gene therapy
• Replacing a mutated gene that causes
disease with a healthy copy of the gene
• Inactivating, or “knocking out” a mutated
gene that is functioning improperly.
• Introducing a new gene into the body to
help fight a disease.
Two new techniques of gene
therapy

RNA interference (post-transcriptional
gene silencing).

Exon skipping.
RNA interference (posttranscriptional gene
silencing):
 Briefly,
double stranded RNA,
homologous to the gene targeted
for suppression, is introduced into
cells where it is cleaved into small
fragments of double stranded
RNA named short interfering
RNAs (siRNA).
These siRNAs decrease
gene expression by:
• Guiding the enzymatic destruction
of the homologous RNA, preventing
translation to active protein.
• Priming RNA polymerase to
synthesis more siRNA, and
resulting in persistent gene
suppression.
• To effectively silence specific genes in
mammalian cells, a short hairpin RNA
(shRNA) was designed.
• These sequences, result in the
transcription of a double stranded RNA
brought together by a hairpin loop
structure.
• These shRNAs effectively mimic siRNA
and result in specific and persistent gene
suppression in mammalian cells.
Exon skipping

The technique allows selected exons to be
deleted from the final protein.

This occur by using short sequences of
RNA that are complementary to exon
recognition sequences or exon splicing
enhancer sequences.

The expressed complementary RNA will
bind to these regions of the gene and
prevent the splicing of intron and exon at
that site. The result of the altered posttranscriptional processing is the removal
of a target exon from the final protein.
Germ line gene
therapy
Definition
• It is gene therapy which is targeted to egg
and sperm cells (germ cells), and would
allow the inserted gene to be passed on
to future generations.
Principle
• Addition of the transferred gene
to the nuclear genome and its
stable transmission to
subsequent generations in a
Mendelian fashion.
Importance

Genes could be "corrected" in the
egg or sperm you are using to
conceive.

The child that results would be
spared certain genetic problems that
might otherwise have occurred.
Disadvantages
 Germline gene transfer might affect germ
cells by making changes that could disrupt
the development of the embryo or fetus in
unexpected ways.
 It is not right to make changes to a germ
line, because some of the people who will be
affected are not even born yet and
therefore cannot give their consent.
Ooplasmic transfer
is a process in which some
cytoplasm from a healthy donor
egg is injected into an egg from
a woman with fertility problems.
This procedure increases the
ability of the recipient egg to be
fertilized and develop into a
healthy embryo.

Mitochondrial DNA may has a role in
this management.

The children born following this
procedure have three genetic
parents, since they carry
mitochondrial DNA from the donor
mother and nuclear DNA from the
mother and father.

This procedure represents the first
instances of germline gene transfer
in humans.
The
Technique of
Ooplasmic
Transfer:
Cytoplasm is
extracted from
the donor's egg
and injected,
with sperm, into
the recipient's
egg, resulting
in ooplasm that
contains
mitochondrial
DNA from both
the donor and
the recipient.
Ooplasmic transfer
A)Two different oocytes 10 minutes after ooplasmic
injection with stained donor ooplasm.
B) Three pronuclear zygot 24 hoursafter ooplasmic
injection with stained donor ooplasm.
Pronuclear microinjection
 Microinjection is technique for
introducing a solution of DNA
into a cell using a fine microcapillary pipette.
 Pronuclear microinjection is an
technique used for gene transfer
at the embryonic stage.
Principle
• A fine glass needle is used to inject a purified
double stranded DNA sequence into the
nucleus of a fertilized mammalian oocyte.
• This process leads to the integration of the
sequence (transgene) into the genome. As a
result, the animal is born with a copy of the
new sequence in every cell.
Disadvantages
It is suggested that embryo gene transfer is
unsafe, as its use results in random
integration of the transgene, a lack of
control of the number of gene copies
inserted, significant rearrangements of host
genetic material, and insertional
mutagenesis.
So, this approach is not applied on humans.
Injecting a vector.
Once a vector has
been incorporated
correctly into the
genome of the
embryonic stem
cells, the cells are
expanded in
culture and
injected into 3.5
day old mouse
blastocysts. The
blastocysts are
injected into the
uterus of a
pregnant female
and the embryos
are allowed to
come to term. Mice
with brown coat
colour are selected
and bred to make
pure knockout
mice.
Somatic Cell Nuclear Transfer (SCNT)

A somatic cell is fused with an enucleated
oocyte.

The nucleus of the somatic cell provides
the genetic information, while the oocyte
provides the nutrients and other energyproducing materials that are necessary for
development of an embryo.

Once fusion has occurred, the cell is
totipotent, and eventually develops into a
blastocyst, at which point the inner cell
mass is isolated. The pluripotent stem cell
line is then established.
• Pluripotent stem cells derived from
SCNT were capable of differentiating
into all cell types, including
gametes.
• Because SCNT involves cloning,
there are many ethical concerns in
using this technique in humans.
• For this reason, experiments of this
nature have only been conducted in
mice.
Ethical issues surrounding
gene therapy
 How can “good” and “bad” uses of gene
therapy be distinguished?
 Who decides which traits are normal
and which constitute a disability or
disorder?
 Will the high costs of gene therapy
make it available only to the wealthy?


Could the widespread use of gene
therapy make society less accepting
of people who are different?
Should people be allowed to use
gene therapy to enhance basic
human traits such as height,
intelligence, or athletic ability?