Gene therapy.ppt

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GENE THERAPY

• M YOUSRY ABDEL-MAWLA,MD
• ZAGAZIG FACULTY of MEDICINE EGYPT

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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
  sequence.

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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.

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DNA replication

• Base excision repair in which deamination
  converts cytosine to uracil and adenine to
  hypoxanthin.
• 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..

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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.

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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

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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

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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

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Gene delivery systems

• Gene delivery systems include viral and non viral
  vectors
• 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.

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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
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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
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Applications of gene therapy

• 1-Gene therapy for systemic diseases via the
  skin
• 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
  .

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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
  lymphoma.
• 4- Congenital hair disorders.
• 5- Wound healing..
• 6- DNA vaccine.
• 7- Genetic pharmacology.
• 8 Pro-drug activation or suicide gene for
  cancer.
• 9 - Nucleic acid agents include antisense
  technology
• (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.

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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 (
  siRNA)

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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).

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MicroRNAs and the skin

• Humans inherit 23 chromosomes from each parent to
  form a diploid genome consisting of 46
  chromosomes.
• 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)

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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

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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.

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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.

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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
  gene

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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.
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MicroRNAs (miRNAs) and short interfering RNAs
(siRNAs)

• They are classes of regulatory small RNA
  molecules, ranging from 18 to 24 nucleotides in
  length,
• 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
  pathway
• 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
  arthritis
• In contrast, miR-125b is down-regulated in
  psoriasis

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• 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
  interferon-
• STAT3 plays a critical role in many biological
  activities, such as cell proliferation,
  migration, homeostasis, inflammation, immune
  regulation and oncogenesis

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SOCS-3 Vs STAT3

• 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

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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.

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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.

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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

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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

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miRNAs and skin cancer

• miRNAs and their key regulators are
• essential for morphogenesis of the skin and hair
  follicles.
• 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)

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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
  expression
• 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

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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
  phenotypes

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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

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• 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
  apoptosis.
• Cylindromatosis tumor suppressor gene, CYLD, is a
  suppressor of NF-kb activation

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• 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
  activation.

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