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.
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 .
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..
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.
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 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 .
Biologic viral vectors. -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
-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 size~4-5 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
-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
-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
-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
Molony murine leukaemia Virus
Limitations Advantages Integration To gemone Type of vectors
Non viral vectors. - 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
- Random integration unless targeted, inefficient DNA transfer,
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 - 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 - Transient gene expression, DNA not integrated into the genome, remain episomal. - simple, relatively efficient, non immunogenic, no mutagenesis. 1-Nacked plasmid DNA Disadvantages Advantages Vector system
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.
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.
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 ( siRNA)
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.
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.
Methods for overcoming the skin barrier 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 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)
Non-coding RNAs (ncRNA)
Few ncRNAs are characterized: Ribosomal RNAs (rRNAs)
Transfer RNAs (tRNAs)
MicroRNAs are small 21–25 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 endogenous—that is, either naturally occurring in the genome or introduced from outside the cell.
MicroRNAs (miRNAs) Functions
MicroRNAs ( miRNAs ) are very small endogenous RNAmolecules about 22–25 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
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 5*end .
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
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 .
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
DNA is packaged among histone proteins which can be modified 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 or targeted for destruction by ubiquitination
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
MicroRNAs in Cutaneous Biology.
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.
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
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
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
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
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 m iR-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 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)
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
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 phenotypes
miR &Kaposi’s 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 apoptosis.
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 activation.