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RNA INTERFERENCE. Pigment enhancing gene. Accidental Discovery. Control. Mex-3 antisense RNA. Mex-3 antisense + sense RNA. Nobel Prize for Medicine-2006 Fire and Mello. mex-3 , highly expressed in C. elegans embryos. RNA Interference.
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Pigment enhancing gene Accidental Discovery
Control Mex-3 antisense RNA Mex-3 antisense + sense RNA Nobel Prize for Medicine-2006Fire and Mello mex-3, highly expressed in C. elegans embryos
RNA Interference The phenomenon where double stranded RNA causes the silencing of genes by targeting complimentary mRNA for degradation. Widely found in eukaryotic species (fungus, plants and animals)
Sources of double stranded RNA • MicroRNAs (miRNAs) • Viruses • Jumping genes
MicroRNAs (miRNAs) • Derived from ~70 nt pre-miRNAs • 21-23 nucleotides (nt) in length • Two base pair overhangs • Transcribed by RNA polymerase II • Do not encode a protein • Many found in the intronic regions of genes
miRNAs as a Therapeutic Tool Every disease caused by activity of one or a few genes • Cancer • Autoimmune diseases • Dominant genetic disorders • Viral infections
siRNA therapy for hypercholestrolemia Synthesis of siRNA for mouse apoB Chemical modification to prevent from degradation Injection in tails of mice Within 24 hours serum LDL reduced by over 50%
siRNA therapy for ALS Define optimum anti-SOD1 siRNA sequences in tissue culture Incorporate sequence in retroviral vector Injection into spinal cord of mutant mice Retardation in onset and progression of ALS
RNAi & Age-related Macular Degeneration (AMD) • Over expression of vascular endothelial growth factor (VEGF) • siRNA against the VEGF gene • Inject directly into the eye • Suppression of VEGF protein • Suppression of angiogenesis in the eye Human clinical trials successful
RNAi: The obstacles • Delivery to the desired cell type, tissue or organ • Stimulation of innate immune response • Suppression of off-targets
Stem Cells Stem cells are • Unspecialized • have the ability to divide and renew themselves indefinitely • can differentiate into one or more specialized cell types
Fertilized egg TOTIPOTENT Types of stem cells Inner cell mass Embryonic stem cells PLURIPOTENT Blastocyst Embryonic germ cells PLURIPOTENT Fetus Adult stem cells MULTIPOTENT or UNIPOTENT
Stem Cell Research Two types of cells • Embryonic stem (ES) cells • Adult stem cells
ES Cells • are derived from the inner mass of a blastocyst • are capable of unlimited cell division • are pluripotent • express the transcription factor Oct-4
Adult stem cells • Generate cells to replace those lost through normal wear and tear, injury or disease • Are identified by the tissue from which they originated. • are found in minute quantities in the bone marrow, blood, cornea, retina, skeletal muscle, liver, skin, brain etc. • Can be made to differentiate into different cells under specific experimental conditions
Potential uses of stem cells • Therapeutic Cloning: Treat human diseases and injuries where the damaged cells or tissues cannot heal or renew themselves • Study basic genetic mechanisms responsible for the processes of development and differentiation. • Test different substances (drugs and chemicals) on stem cells.
Advantages and Disadvantages of Embryonic and Adult Stem Cells
Focus of Stem Cell Research • determining precisely how stem cells remain unspecialized and self renewing for many years • identifying the signals (internal as well as external) that cause stem cells to become specialized cells
Induced Pluripotent Stem Cells (iPSCs) 2006: Adult mouse fibroblasts converted to pluripotent cells (iPS cells) on injection with genes coding for four transcription factors (Oct-3/4, SOX2, c-Myc, and Klf4). 2007: iPS cells could give rise to all cell types and grown into baby mice when injected into a mouse blastocyst 2008: Skin cells from 80 year old ALS patient converted to iPS cells
stem cell therapy… Success stories!! July 2011 November 2008