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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 .
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
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
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 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
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.
Antisense therapy is a form of treatment for genetic disorders or infections. When the genetic sequence of a particular gene is known to be causative of a particular disease, it is possible to synthesize a strand of nucleic acid (DNA, RNA or a chemical analogue) that will bind to the messenger RNA (mRNA) produced by that gene and inactivate it, effectively turning that gene "off".
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).
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
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 (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
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.
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
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-
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
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