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Gene therapy.ppt


The medical application of gene therapy in dermatology.ppt – PowerPoint PPT presentation

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Title: Gene therapy.ppt


DNA replication
  • The DNA in the chromosomes is replicated during a
    period of interphase called S-phase of cell cycle
    which stands for synthesis of DNA.
  • strands could come apart and each separated
    strand serve as template for the synthesis of a
    new partner strand complementary in nucleotide

DNA repair system
  • Mismatch repair a pair of non-hydrogen bonded
    bases (e.g. G-----T) within a helix is recognized
    as aberrant and a polynucleotide segment of
    daughter strand is excised, thereby removing one
    member of the unmatched pair.
  • Nucleotide excision repair lesions that distort
    the double helix as a thymine dimer can also be
    repaired by the excision of a short stretch of
    nucleotides including the lesion, followed by its
    correct replacement , the opposite strand serving
    as the template.

DNA replication
  • Base excision repair in which deamination
    converts cytosine to uracil and adenine to
  • DNA glycosylases recognize the abnormal bases and
    hydrolyse them of, leaving apurin or apyrimidine
    sites in which the deoxyribose has no base
    attached to it..

Gene therapy
  • Gene therapy is divided into
  • Germ line gene therapy
  • Somatic gene therapy.
  • Germ line gene therapy the therapeutic gene
    modification is introduced into all cells of the
    body or a subset of cells including germ cells.
  • Somatic gene therapy the genetic modification is
    restricted exclusively to somatic cells with no
    effect on the germ line.

Gene Delivery
  • In vivo delivery direct introduction of genetic
    material into the skin of the patient
  • Treatment of metastatic malignant
    melanoma. Skin tumors were injected directly with
    plasmid DNA designed to express the human
    leucocyte antigen (HLA) class I gene, B7, which
    is chosen to be mismatched with the patient's HLA
    type 9. In vivo gene therapy may more accurately
    represent the actual interactions between the
    skin and surrounding tissues

Ideal in vivo gene delivery system
  • High efficiency of uptake of the therapeutic gene
    by the target cells, transportation of the
    therapeutic gene to the nucleus of the target
    cell with minimal of intracellular degradation
    and sustained expression of the therapeutic gene
    at a level that alleviates the condition

Ex vivo delivery
  • This involves removal of a skin sample from the
    patient, followed by propagation of skin cells
    (eg stem cells)in culture, introduction of
    genetic material into the cultured cells, and
    return of the epithelailized genetically
    engineered cells in the form of a skin graft back
    to the patient .Ex ex vivogene therapy using stem
    cell in treatening of epidermolysis bullosa

Gene delivery systems
  • Gene delivery systems include viral and non viral
  • The ideal vector as a mean of delivering genes to
    human cells and tissues is that vector which
    delivers genes with high efficiency into the
    proper tissue.
  • Ideal vector should either remain in a stable
    extra-chromosomal state or to have the ability
    to target a specific site within the genome.

Biologic viral vectors.
Limitations Advantages Integration To gemone Type of vectors
-Does not infect non dividing terminally differentiated cells, may be oncogenic optimal insert size 5-7 kb. -Wide host range high efficiency transduction of dividing cells efficient expression of foreign gene product stable integration infects only once and does not replicate in vivo. Yes - Retrovirus Molony murine leukaemia Virus
-Wide host range stable transduction of dividing and non dividing terminally differentiated cells with long term expression nonpathogenic lack of expression of viral proteins. Yes Leintvirus (HIV)
-Expression of viral proteins results in toxic reaction and inflammation carcinogenic low efficiency in dividing cells, short-term expression, insert size only 7-11 kb. -Transduction of non dividing cells with high efficiency wide host rang high viral titer and high expression levels newly developed gutless vectors have insert size as large as 30 kb. No Adenovirus
-Limited transduction efficiency that depends on helper viral functions, although in newer systems helper virus not needed, low efficiency of integration to genome small insert size4-5 kb. -Transduction of dividing and non dividing cells, all viral coding sequences can be deleted except those required for transduction non immunogenic and nonpathogenic long-term expression of transgene specific integration site (some forms). Yes Adeno-associated virus
-Short-term expression spreading of the infection to surrounding cell populations new engineered vectors are avirulent in surrounding terminally differentiated cells immunogenic. - Transduction of neurons and glial cells, wide host range large insert size up to 30 kb efficient infection. No Herpes simplex virus
Non viral vectors.
Disadvantages Advantages Vector system
- Transient gene expression, DNA not integrated into the genome, remain episomal. - simple, relatively efficient, non immunogenic, no mutagenesis. 1-Nacked plasmid DNA
- Random integration, inefficient DNA transfer. -- -- Unstable, remain episomal, poor gene expression. -Unstable, remain episomal, poor or gene expression. - Easy to use. - Non infectious, non immunogenic, effective for in vivo gene transfer, can carry large DNA fragments. - Targeted delivery, can carry large fragments of DNA. 2- Chemical vectors a-Calcium phosphate b-Cationic liposomes (lipoplex) c-Polylysine-DNA complexes
-- - Random integration unless targeted, inefficient DNA transfer, Superficial burn. No integration of DNA, transient gene expression. - Ineffective in large surface area. - Easy to use, safe, many cell type and different applications. - Cell receptor independent, delivers genes to different tissues. - used in vaccine protocols, effective in localized area, increase gene expression, painless, no bleeding. 3- Physical methods a- Electroporation b-Gene gun approa-ch c-Microneedle injection
- Still in developmental stages. - Stable, non-infectious, can carry large fragments of DNA, non immunogenic, no integration into the genome. 4-Biologic non viral vectors human artificial chromosomes
Applications of gene therapy
  • 1-Gene therapy for systemic diseases via the
  • a- Skin acts as a secretory organ e.g.
    in haemophilia and growth hormone deficiency.
  • b- Skin acts as a metabolic sink or
    a bioreactor in the following metabolic disorders
    e.g. - Adenine deaminase deficiency ,Ornithine
    aminotransferase deficiency or hypercholestrelemia

Applications of gene therapy
  • 2- Inhereted skin diseases e.g. xeroderma
    pigmentosum, ichthyoses (X-linked and lamellar
    types), epidermolysis bullosa (junctional and
    dystrophic types).
  • 3- Skin malignancies e.g. malignant
    metastatic melanoma and cutaneous T-cell
  • 4- Congenital hair disorders.
  • 5- Wound healing..
  • 6- DNA vaccine.
  • 7- Genetic pharmacology.
  • 8 Pro-drug activation or suicide gene for
  • 9 - Nucleic acid agents include antisense
  • (RNA and oligonucleotides), ribozymes,
    and splicisome-mediated RNA trans-splicing.
  • 10- Nucleic acid pharmaceuticals.

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Antisense Gene Therapy (AS-ODNs)
  • The discovery of antisense oligonucleotides
    (AS-ODNs) and small interfering RNA( siRNA) has
    opened wide perspectives in therapeutics for the
    treatment of cancer, infectious and inflammatory
    diseases or to block cell proliferation and
    diseases caused thereby.

Small interfering RNA ( siRNA)
  • Gene expression could be inhibited by the
    introduction of double-stranded RNA with sequence
    complementarity to the gene being targeted, a
    mechanism that was named RNA interference (

Short interfering RNA(siRNA)
  • long, double-stranded RNAs are introduced into a
    cell, they become diced into short,
    double-stranded, 21-nt RNAs containing 2-nt 39
    overhangs, known as
  • short interfering RNA (siRNA).
  • The siRNA then guide cellular machinery to target
    and degrade mRNA with a similar sequence.

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Antisense oligonucleotoids(AS-ODNs)
  • Synthetic single-stranded DNA fragments that bind
    to specific intracellular messenger RNA strands
    (mRNA) forming a short double helix. They
    consist of short sequences, composed of 13 to
    about 25 nucleotides, which are complementary to
    mRNA strands in a region of a sequence designed
    as sense strand.
  • By binding to the mRNA molecules, AS-ODNs are
    shown to stop translation of the mRNA, and hence
    protein synthesis expressed by the targeted gene.

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Methods for overcoming the skinbarrier against
gene delivery
  • To achieve more efficient cutaneous gene
    delivery,removal of the horny layer is thought to
    be the best way to disrupt the barrier of the
    skin. Tape-stripping using adhesive tape may be
    used to remove the horny layer.
  • Several technological advances have been made in
    overcoming this barrier electroporation,
    sonophoresis iontophoresis and chemical
    penetration enhancers (CPEs).

MicroRNAs and the skin
  • Humans inherit 23 chromosomes from each parent to
    form a diploid genome consisting of 46
  • The majority of the genome actually consists of
    non-coding genes and regions. For a long time
  • The majority of the DNA in our genomes, initially
    labeled as unnecessary
  • Useless DNA, is actively transcribed into
    functional primary RNA ,it transcripts or
    non-coding RNAs (ncRNA)

Non-coding RNAs (ncRNA)
  • Few ncRNAs are characterized
    Ribosomal RNAs (rRNAs)
  • Transfer RNAs (tRNAs)
  • MicroRNAs are small 2125 nt RNA molecules that
    are essential regulators of a wide range of
    cellular processes

Micro RNAs(MiRNAs)vs siRNAs
  • MiRNAs refer to small RNAs produced naturally
    from the human genome, and have diverse and
    widespread roles.
  • They are generated by transcribing a single RNA
  • siRNAs can be either exogenous or endogenousthat
    is, either naturally occurring in the genome or
    introduced from outside the cell.

MicroRNAs (miRNAs) Functions
  • MicroRNAs (miRNAs) are very small endogenous
    RNAmolecules about 2225 nucleotides in length,
    capable of post-transcriptional gene regulation.
  • miRNAs bind to their target messenger RNAs
    (mRNAs), leading to cleavage or suppression of
    target mRNA translation

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The Biogenesis of miRNAs
  • miRNAs are transcribed by RNA polymerase II in
    mammalian cells
  • The primary miRNA transcript (pri-miRNA) is
    usually several kilobases long, poladenylated at
    its 3 end and capped with a 7-
    methylguanosine cap at its 5end .
  • The intranuclear RNase III enzyme then cleaves
    the pri-miRNA, which may contain multiple miRNA,
    into several precursor miRNAs (premiRNAs). DGCR8
    (DiGeorge syndrome critical region gene 8/) is
    essential for RNASE activity

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Molecular biology
  • Heritable genetic information is contained in
    DNA, which can be replicated and passed to
    daughter cells.
  • DNA is transcribed to RNA, transported to the
    nucleus, and translated into proteins. The
    identification of reverse transcriptase
    demonstrated that RNA can also be converted back
    into DNA.

Molecular biology
  • Gene activity is regulated on many levels.
    Representative mechanisms of gene regulation are
    shown at the DNA, RNA, and protein levels. The
    rate of gene transcription can be affected by the
    quantity of transcription factors (green circles)
    that are locally available to interact with the

Molecular biology
  • DNA is packaged among histone proteins (spheres),
    which can be modified (red octagons) in a way to
    package DNA more tightly and make it less
    accessible to transcription factors.
  • On the RNA level, the stability of a transcript
    can determine how long it persists in the cell
    and how much protein can be made. At the protein
    level, proteins can be switched to active form by
    chemical modifications, such as phosphorylation
    (gold star) or targeted for destruction by
    ubiquitination (pink hexagons).
    Polyubiquitination causes proteins to be ferried
    to the proteasome, which degrades proteins into
    short amino acids.
  • MicroRNAs function at the level of altering RNA
    stability, as well as by affecting the rate at
    which RNAs are translated into proteins

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MicroRNAs in Cutaneous Biology.
MicroRNAs (miRNAs) and short interfering RNAs
  • They are classes of regulatory small RNA
    molecules, ranging from 18 to 24 nucleotides in
  • Their roles in development and disease are
    becoming increasingly recognized.
  • They function by altering the stability or
    translational efficiency of messenger RNAs
    (mRNAs) with which they share sequence
    complementarity, and are predicted to affect up
    to onethird of all human genes.

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miRNAs and psoriasis
  • Tumor necrosis factor (TNF)-a is a
    proinflammatory cytokine shown to play an
    important role in the pathogenesis of psoriasis
  • Three different miRNAs have thus far been
    associated with this skin disease and linked to
    both innate immune responses and the TNF-a
  • miR-203 was the first miRNA found to be
    significantly overexpressed in psoriasis patients
  • Up-regulation of miR-203 leads to
    down-regulation of suppressor of cytokine
    signaling-3 (SOCS-3) expression in psoriatic skin
  • miR-146a is overexpressed in many psoriatic
    skin lesions and patients with rheumatoid
  • In contrast, miR-125b is down-regulated in

  • SOCS-3 is an,inhibitor of the signal transducer
    and activator of transcription 3 (STAT3) pathway
    , which is widely expressed and activated by
    various growth-regulating signals and
    inflammatory cytokines such as interleukin-6 or
  • STAT3 plays a critical role in many biological
    activities, such as cell proliferation,
    migration, homeostasis, inflammation, immune
    regulation and oncogenesis

  • STAT3 has been shown to be constitutively
    activated in epidermal keratinocytes of human
    psoriatic lesions
  • Inhibition of STAT3 has drastically improved
    clinical prognoses in psoriatic patients

miR-146a and psoriasis
  • miR-146a targets, TNF receptor-associated factor
    6(TRAF6) and IL-1R-associated kinase (IRAK)
    which are all involved in the TNF-a pathway,
    which contributes to psoriatic skin inflammation.

miRNAs and wound healing
  • Wound healing can be divided into four phases
    inflammatory, proliferative, fibroplasia
    maturation, and remodeling phase.
  • Platelets secrete various cytokines, including
    platelet-derived growth factor (PDGF), platelet
    factor IV and transforming growth factor beta
  • (TGF-b)
  • miR-140 has been shown to have a modulating
    effect on PDGF receptor a.

miRNAs and wound healing
  • Polymorphonuclear leukocytes and macrophages
    migrate to the wound site and release a variety
    of chemotactic factors such as fibroblast growth
    factor (FGF), TGF-band TGF-a, plasma-activated
    complements C3a and C5a, interleukin-
  • 1 (IL-1), tumor necrosis factor (TNF) and PDGF.
  • TNF-a is regulated by miR-146a targets

miRNAs and angiogenesis
  • The role of miRNAs in angiogenesis has been the
    subject of numerous studies
  • overexpression of miR-221 and miR-222 indirectly
    reduces the expression of endothelial nitric
    oxide synthase (eNOS), which is essential for
    many endothelial cell functions

miRNAs and skin cancer
  • miRNAs and their key regulators are
  • essential for morphogenesis of the skin and hair
  • It is thus expected that a disruption of miRNA
    expression can be observed in
  • various malignant skin lesions.
  • miR-218-1 is a tumor suppressor inactivated in
    breast, lung and colorectal cancers. It is
    located within the tumor suppressor gene SLIT2
    (human homologue of Drosophila Slit2)

miR and Melanoma
  • miR-137 modulates expression of
    microphthalmia-associated transcription factor
    (MITF), which is a major regulator of melanocyte
    growth, maturation, apoptosis and pigmentation
  • miR-221 miR-222,indirectly regulate MITF
  • Ultraviolet radiation-induced sun tanning occurs
    through keratinocyte expression of a-melanocyte
    stimulating hormone (a-MSH), which then leads to
    melanocyte MITF expression.
  • MITF induction protects the skin from DNA damage.
  • Expression of melanoma inhibitor of apoptosis
    (MLIAP) in melanoma cells is MITF-dependent

miR-221 miR-222 in Melanoma
  • miR-221 miR-222 primarily control melanoma
    progression through down-regulation of
    cyclin-dependent kinase inhibitor 1b
    (p27Kip1/CDKN1B) and c-KIT receptor, both of
    which play critical roles in melanocyte
    physiology and favor induction of malignant

miR Kaposis sarcoma
  • A form of skin cancer associated with herpes
  • virus (KSHV), has been identified as a causative
    agent of several diseases such as primary
    effusion lymphoma (PEL).
  • Human miR-155 shares several targets and binding
    sites such as the transcriptional regulators
    BACH-1, FOS and the proapoptotic
  • effector LDOC-1 with viral miR-k12-11.
  • The possibility that mir-k12- 11 may play a role
    in tumorgenesis by interfering in the network of
    transcripts that are regulated by miR155
    indicates a possible link between viral and
    non-viral tumorigenesis

  • TNF-a signaling has been closely tied with tumor
    formation, and its activation upregulates the
    nuclear transcription factor nuclear factor kappa
    B (NF-kb).
  • NF-kb is broadly involved with inflammatory
    responses, immunity, and protection against
  • Cylindromatosis tumor suppressor gene, CYLD, is a
    suppressor of NF-kb activation

  • CYLD functions as a deubiquitinating enzyme,
    responsible for removing ubiquitin groups from
    specific proteins.
  • Ubiquitination of TNF-receptor related factor
    (TRAF ) activates its association with the
    inhibitor of kappa-beta kinase complex (Ikb),
    leading to upregulation of NF-kb and prevention
    of apoptosis
  • In a normal state, CYLD functions to block TRAF
    ubiquitination, and protects against NF-kb

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