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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