Gene Therapy & Ribozymes

Mohammed Shahid Khan Bilal Hassn Kirmani Shahrukh Babar Zulkifal Yousaf Zuhaib Anwar Usman Aziz Adil Bhatti 2

Overview Gene therapy is the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease Correction of defective genes that are responsible for disease development The technology is still in its infancy 3

APPROACHES OF GENE THERAPY Replacing a mutated gene Inactivating, or “knocking out Introducing a new gene 4

TYPES OF GENE THERAPY Germ line gene therapy – germ cells, i.e., sperm or eggs are modified by the introduction of functional genes, which are integrated into their genomes – jurisdictions prohibit this for application in human beings Somatic gene therapy – genes are transferred into the somatic cells of a patient – will not be inherited by the patient's offspring or later generations. – is viewed as a more conservative, safer approach because it affects only the targeted cells in the patient – effects of somatic cell therapy are short-lived – somatic cell gene therapy is appropriate and acceptable for many disorders – Clinicians can even perform this therapy in utero 5

CATEGORIES OF SOMATIC GENE THERAPY EX VIVO TECHNIQUE – Cells are modified outside the body and then transplanted back in again – The cells are exposed to the virus that is carrying the desired gene then returned to the patient by injection into a vein. IN VIVO TECHNIQUE – Genes are changed in cells still in the body 6

APPLICATIONS Replace missing or defective genes Deliver genes that speed the destruction of cancer cells Supply genes that cause cancer cells to revert back to normal cells Deliver bacterial or viral genes as a form of vaccination Provide genes that promote or impede the growth of new tissue Deliver genes that stimulate the healing of damaged tissue 7

Delivery Genes can be carried into cells by viruses In virus carriers, the DNA coding for some or all of the normal genes of the virus to be used as a carrier are removed and replaced with a treatment gene They are innocuous Genes can also be delivered within tiny synthetic "envelopes" of fat molecules Genes can also gain entrance into cells through electroporation 8

Route of Administration The choice of route for gene therapy depends on the tissue to be treated and the mechanism by which the therapeutic gene exerts its effect 9

PROBLEMS Short-lived nature of gene therapy Immune response Problems with viral vectors Multigene disorders Chance of inducing a tumor (insertional mutagenesis) 10

APPLICATIONS Bionic chip – A new "bionic chip" has been developed to help gene therapists using electroporation to slip fragments of DNA into cells – It contains a single living cell embedded in a tiny silicon circuit. The cell acts as a diode, or electrical gate 11

RIBOZYMES Before ribozymes were discovered in the early 1980s, all enzymes were thought to be proteins The latest additions include : – ribonuclease P, – group I intron structures, – ribosome (the peptidyl transferase appears to be a ribozyme) Several smaller ribozymes, including: – Diels–Alderase, – glmS ribozyme – a new hammerhead ribozyme structure 12

Introduction Not all ribozymes are metalloenzymes Ribozymes are enzymes whose catalytic centers are composed entirely of RNA Acid-base catalysis appears to be a catalytic strategy so fundamental that it occurs in both protein and RNA enzymes Several of the small self-cleaving RNAs as a consequence do not strictly require divalent metal ions for catalysis 13

Ribonuclease P First true RNA enzyme identified RNA–protein complex 14

Group I intron The folds of the various group I introns are quite similar Azoarcus structure, In which both exons were present Tetrahymena group I intron structure, in which guanosine and a metal ion are present in the active site 15

A Diels–Alderase ribozyme Diels–Alderase is a catalytic antibody The structure of a Diels–Alder ribozyme is in both the unbound and enzyme–product complex states 16

The glmS ribozyme First, it is a ribozyme that is also a riboswitch Second, the regulatory effector of the ribozyme participates in the acid- base catalysis of RNA self-cleavage GlcN6P production is regulated in many Gram-positive bacteria 17

The hammerhead ribozyme The best experimentally characterized RNA enzyme Motif consists of three base-paired stems flanking a central core of 15 conserved nucleotides Most of these conserved bases cannot form conventional Watson- Crick base pairs Substitution results in diminished catalytic activity 18

FINI 19