Gene Therapy & Ribozymes

2

Mohammed Shahid KhanBilal Hassn Kirmani

Shahrukh BabarZulkifal YousafZuhaib Anwar

Usman AzizAdil Bhatti

3

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

4

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

5

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

6

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

7

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

8

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

9

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

10

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

11

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

12

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

13

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

14

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

15

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

16

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

17

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

18

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

19

FINI