PATENT PROJECT FINAL PRESENTATION TITLE : USE OF PROSTAGLANDIN E 2  RECEPTOR GENE(EP2) FOR RELAXATION OF CILIARY MUSCLES AND THEREBY HELP IN THE TREATMENT OF FUNCTIONAL MYOPIA -SACHIN JAMBAWALIKAR

ABSTRACT : A NOVEL APPROACH TO USE GENE THERAPY TO TRANSFECT THE CILIARY MUSCLE CELLS WITH PROSTAGLANDIN 2  RECEPTOR GENE.THE EXPRESSION OF THIS GENE WILL INDUCE A SERIES OF EVENTS WHICH INCLUDE THE CYCLIC -AMP(SECOND MESSENGER)PATHWAY AND CAUSE THE RELAXATION OF THE CILIARY MUSCLE IN THE FUNCTIONAL MYOPIC PATIENTS.

BACKGROUND: MYOPIA: ITS DEFINED AS THE STATE WHERE THE RAYS OF LIGHT EMERGING FROM A DISTANT OBJECT FOCUS AT A POINT IN FRONT OF THE RETINA THEREBY PRODUCING A BLURRED IMAGE ON THE RETINA. TYPES: STRUCTURAL MYOPIA: OVER ELONGATION OF THE EYE BALL CAUSES STRUCTURAL MYOPIA FUNCTIONAL MYOPIA: CILIARY MUSCLES ARE WEAKENED AND COMPLETE RELAXATION DOESNOT TAKE PLACE THEREBY MAKING THE LENS MORE CURVED.

AVAILABLE TREATMENT OPTIONS : 1)OPTICAL CORRECTION 2)MEDICAL(PHARMACEUTICAL): CYCLOPLEGIC AGENTS 3)VISION THERAPY 4)ORTHOKERATOLOGY 5)REFRACTIVE SURGERY 6)FOOD AND NEUTRITION THERAPY

HYPOTHESIS AND ASSUMPTION ON WHICH THIS INVENTION IS BASED. THE FUNCTIONAL MYOPIA WHEN SEEN FROM THE MOLECULAR LEVEL MIGHT BE CAUSED DUE TO A PROBLEM IN THE CELL SIGNALLING MECHANISM. IT MIGHT BE CAUSED DUE TO DEGRADATION OR PERMANENT BLOCKING OF RECEPTORS SUCH AS PROSTAGLANDIN 2E ALPHA RECEPTOR. A RESULT OF THIS IS INCOMPLETE RELAXATION OF CILIARY MUSCLE THIS CAUSES THE LENS TO REMAIN BULGED DUE TO IMPROPER ACCOMODATION AND THE PERSON BECOMES MYOPIC.

DETAILED EXPLAINATION OF THE INVENTION: 1) VIRUS VECTOR USED FOR GENE DELIVERY. 2)GENE DELIVERY PROCEDURE 3)PGE2 AND EP2 RECEPTOR 4)PGE2 INITIATED CYCLIC AMP PATHWAY AND RELAXATION OF CILIARY MUSCLE

VIRUS VECTOR : -THE VIRUS VECTOR USED WAS HSV-1,HrR3. -THE VIRUS GENETIC SEQUENCE WAS ALTERED BY ADDITION OF EP2 RECEPTOR GENE. -HSV1 VIRUS IS USED AS COMPARED TO ADENOVIRUS AS HSV1 HAS HIGH EFFICIENCY IN GENE DELIVERY TO THE NERVOUS SYSTEM. COMPARED TO ADENO VIRUS -HSV1 IS KNOWN TO HAVE MUCH HIGHER CAPACITY FOR ACCOMODATING MULTIPLE THERAUPATIC GENES.

GENE DELIVERY: -A 30 GAUGE NEEDLE WAS THREADED THROUGH THE TEMPORAL CORNEA OF ANASTHETIZED PATIENT. -THE EP2 RECEPTOR GENE WITH ITS VECTOR IS INJECTED IN THE ANTERIOR CHAMBER. -THE HSV-1 VIRUS TRANSFECTS THE GENE INTO THE MUSCLE CELLS OF THE CILIARY BODY.

PGE2-ALPHA AND EP2 RECEPTORS : -PROSTAGLANDINS ARE A GROUP OF EICOSANOID. -THESE SIGNALLING MOLECULES OFTEN ACT IN AN AUTOCRINE MODE IN MATURE MAMMALS. -THEY ARE FATTYACID DERIVATIVES MADE BY CELLS IN ALL MAMMALIAN TISSUE. -THEY ARE MADE FROM PRECURSORS(MAINLY ARACHIDONIC ACID)WITH 20CARBON FATTY ACID CHAINS AND ATLEAST THREE DOUBLE BONDS. -PROSTAGLANDIN ARE A PRODUCT OF CYCLOOXYGENASE -CATALYSED METABOLISM OF ARACHIDONIC ACID.

-EP2 RECEPTOR IS A G-PROTIEN LINKED RECEPTOR. -IT IS A TRANSMEMBRANE PROTEIN WITH 7 PASSES THROUGH THE PLASMA MEMBRANE. -THE EP2 RECEPTOR ACTS INDIRECTLY TO REGULATE THE ACTIVITY OF A SEPARATE PLASMA MEMBRANE BOUND TARGET PROTEIN WHICH IS THE CALCIUM ION CHANNEL.

PGE2 INTIATED CYCLIC AMP PATHWAY AND RELAXATION OF CILIARY MUSCLE

STEPS: 1) THE EP2 RECEPTOR GENE ON EXPRESSION PRODUCES THE EP2 RECEPTOR PROTEIN. 2) THIS PROTEIN WILL MOVE TOWARDS THE PLASMA MEMBRANE AND WILL FORM A SEVEN PASS TRANS MEMBRANE PROTEIN. 3)WHEN THE LIGAND (PGE)RELEASED BY NEIGHBOURING CELL ATTACHES TO EP2 RECEPTOR IT CAUSES A CONFORMATIONAL CHANGE IN THE  - SUBUNIT OF THE G PROTEIN. 4)THIS WILL SIGNAL ADENYLYL CYCLASE(AC) A MEMBRANE BOUND INTRACELLULAR ENZYME WHICH CATLYZES THE CONVERSION OF ATP TO CYCLIC AMP.

5) PRODUCTION OF CYCLIC AMP WILL ACTIVATE CYCLIC AMP DEPENDENT PROTEI KINASE(PKA). 6)PKA WILL PHOSPHORYLATE SEVERAL DIFERENT INTRACELLULAR PROTEINS THAT ONCE ACTIVATED LEAD TO MUSCLE RELAXATION BY OPENING OF THE CALCIUM CHANNEL AND A SERIES OF OTHER EVENTS THERE BY CAUSING COMPLETE REPOLARIZATION OF THE CELL.

CLAIMS : 1 )EP2 ALPHA RECEPTOR GENE IS TRANSFECTED INTO THE CILIARY MUSCLE CELLS.THE EXPRESSION OF THIS GENE WILL CAUSE RELEASE OF PROTEIN WHICH ACTS AS A G PROTEIN LINKED RECEPTORAND ON INTERACTION WITH PGE2 LIGAND HELPS IN RELAXATION OF THE CILIARY MUSCLE BY GOING THROUGH THE CYCLIC AMP PATHWAY.THIS CAN BE USED FOR THE TREATMENT OF FUNCTIONAL MYOPIA. 2)THE METHOD OF CLAIM 1 WHERE IN THE VIRUS VECTOR CAN BE RETROVIRUS,ADENOVIRUS,ADENOASSOCIATED VIRUS(AAV),VACCINIA VIRUS AND POLIO VIRUS. 3)THE METHOD IN CLAIM 1 WHERE IN THE ARE OF TREATMENT IS GLAUCOMA.

4) THE METHOD OF CLAIM 1 WHERE THE RECEPTOR GENE CAN BE PROSTAGLANDIN 2FALPHA,2A ALPHA,2B ALPHA,2C ALPHA AND 2D ALPHA. 5)THE METHOD OF CLAIM 1 WHERE IN THE TRANS FECTED GENE IS ADRENERGIC BETA2 RECEPTOR GENE. 6)NITRIC OXIDE SYNTHASE GENE CAN ALSO BE DELIVERED BY THE METHOD 1.THIS WILL PRODUCE NITRIC OXIDE.NO WILL PASS THROUGH THE PLASMA MEMBRANE OF THE TARGET CELLS AND ACTIVATE INTRACELLULAR ENZYMES USUALLY GUANYLYL CYCLASE WHICH WILL PRODUCE CYCLIC GMP. THIS IN TURN PRODUCES PKG WHICH PRODUCES RELAXATION AS IN THE CYCLIC AMP PATHWAY.