RNA INTERFERENCE

Accidental Discovery Pigment enhancing gene

Nobel Prize for Medicine-2006 Fire and Mello mex-3, highly expressed in C. elegans embryos Mex-3 antisense RNA Mex-3 antisense + sense RNA Control

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)

 MicroRNAs (miRNAs)  Viruses  Jumping genes Sources of double stranded RNA

MicroRNAs (miRNAs)  Derived from ~70 nt pre-miRNAs  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

RNA INTERFERENCE

Two Modes of RNA Interference

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 CELL THERAPY

Stem Cells Unspecialized have the ability to divide and renew themselves indefinitely can differentiate into one or more specialized cell types Stem cells are

Growth pattern of a stem cell

Types of stem cells Fertilized egg TOTIPOTENT Blastocyst Embryonic stem cells PLURIPOTENT Inner cell mass Fetus Embryonic germ cells PLURIPOTENT 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.

THERAPEUTIC CLONING

REPRODUCTIVE CLONING

Embryonic Stem CellsAdult Stem Cells “Pluripotent” (can become any cell) “Multipotent” (“can become many but not any”) Stable. Can undergo many cell divisions Less Stable. Capacity for self-renewal is limited Easy to obtain but blastocyst is destroyed Difficult to isolate in adult tissue Possibility of rejection??Host rejection minimized Advantages and Disadvantages of Embryonic and Adult Stem Cells

Potential diseases treatable by stem cells Cell/Tissue typeDisease treatment NeuralParkinson disease Spinal cord injuries SkinBurn victims CardiacRepair of damage associated with heart attacks CartilageRepair of joints damaged by injury or arthritis Pancreatic B islet cellsDiabetes

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

Stem cells therapy: Ethical considerations

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!! November 2008 July 2011