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Gene Therapy

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Title: Gene Therapy


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Gene Therapy
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Presented by
  • Professor
  • Mohamed Abd Ellatif
  • Professor of Medical Biochemistry
  • And Molecular Biology
  • Faculty of Medicine, Mansoura University
  • Mansoura, Egypt

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Definition of Gene therapyGene therapy is an
experimental technique that uses genes to treat
or prevent disease.
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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.

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Principle
  • Introduction of genetic material into cells to
    compensate for abnormal genes or to make a
    beneficial protein.

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Two approaches for delivering genetic material
exist
  • In vivo gene therapy
  • Ex vivo gene therapy

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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.

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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.

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Vectors used in gene therapy
  • Viruses
  • Non-viruses

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Viruses used are
  • Retroviruses
  • Adenoviruses
  • Adeno-associated viruses
  • Lentivirus

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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 long-term
    gene expression.

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Non-viral vectors
  • Direct introduction of therapeutic DNA into
    target cells.
  • Creation of an artificial lipid sphere with an
    aqueous core (liposome).

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  • Chemically linking the DNA to a molecule that
    will bind to special cell receptors.
  • Human artificial chromosomes (HACs)

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Liposome
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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.

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Size
  • liposomes are typically 5-10 µm in diameter with
    the phosopholipid bilayer about 3 nm thick

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Mechanism of formation
  • self-assembly process that is driven by the
    amphipathic nature of phospholipid molecules

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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.

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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. 

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  • Methods for Enhancing The Efficiency of
  • Liposome-Based Transfection

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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

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To improve translocation across the nuclear
membrane
  • nuclear localization signals (NLS) are used
  • classical NLS
  • non-classical M9 NLS

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Artificial chromosomes
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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.

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  • 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.

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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.

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  • 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.

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  • Disadvantages of in vivo gene therapy

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  • The introduced gene will integrate in the genome
    and cause interruption or disruption of other
    gene functions, causing mutation and possibly
    disease.

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Ex vivo gene therapy
  • Approach

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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.
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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.

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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.

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  • 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.

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  • Currently, autologous primary cell cultures are
    used.
  • Autologous means that
  • the donor and
  • recipient organism
  • are the same

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The advantage of using primary, autologous cell
cultures include
  • Lack of antigenicity.
  • Decreased risk of malignant transformation.

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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.

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  • 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.

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Stem cell ex vivo gene therapy
  • Bone marrow derived stem cell.
  • Hepatocytes.
  • CNS stem cells
  • Fetal derived stem cells.

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Human embryo at 4 cell stage
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Stem cells used in treatment of
neuro-degenerative diseases (Infarction)
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Heamobiotic Stem cell
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Fetal Stem Cells
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Blastocyte
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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.

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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.

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Disadvantages
  • Low viral transfection efficiency.
  • Technical difficulties in isolating, culturing,
    and maintaining these cells.

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  • How safety is the gene therapy

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  • 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.

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Factors have kept gene therapy from becoming an
effective treatment for genetic disease
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  • Short-lived nature of gene therapy
  • Immune response
  • Problems with viral vectors as toxicity, immune
    responses, gene control, targeting tissues and
    reactivation of virus.

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  • Lack of viral specificity
  • Multigene disorders as Alzheimers disease, DM
    and heart disease.
  • Inefficient gene transfer

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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.

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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.

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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 Parkinsons disease
  • RNA interference or gene silencing may be a new
    way to treat Huntington's.

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  • New gene therapy approach repairs errors in
    messenger RNA derived from defective genes.
  • Gene therapy for treating children with X-SCID
    (sever combined immunodeficiency) or the "bubble
    boy" disease is stopped when the treatment causes
    leukemia in one of the patients.

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  • Creation of tiny liposomes 25 nanometers (the
    already Known is 5-10 µm) across that can carry
    therapeutic DNA through pores in the nuclear
    membrane.
  • Sickle cell is successfully treated in mice.

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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.

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Two new techniques of gene therapy
  • RNA interference (post-transcriptional gene
    silencing).
  • Exon skipping.

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RNA interference (post-transcriptional 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).

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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.

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  • 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.

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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 post-transcriptional
    processing is the removal of a target exon from
    the final protein.

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Germ line gene therapy
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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.

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Principle
  • Addition of the transferred gene to the nuclear
    genome and its stable transmission to subsequent
    generations in a Mendelian fashion.

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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.

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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.

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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.

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  • 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.

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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.
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Two different oocytes 10 minutes after ooplasmic
injection with stained donor ooplasm.B) Three
pronuclear zygot 24 hoursafter ooplasmic
injection with stained donor ooplasm.
Ooplasmic transfer
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Pronuclear microinjection
  • Microinjection is technique for introducing a
    solution of DNA into a cell using a fine micro-
    capillary pipette.
  • Pronuclear microinjection is an technique used
    for gene transfer at the embryonic stage.

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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.

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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.

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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.
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Pronuclear microinjection Genetic material is
injected directly into the fertilized egg which
is then implanted back into a mouse and allowed
to come to term.
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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 energy-producing
    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.

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  • 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. 

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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?

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  • 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?

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