Gene therapy for bone engineering

Gene therapy for bone engineering SUBHADEEP DAS DEPARTMENT OF ZOOLOGY UNIVERSITY OF CALCUTTA 4TH SEM , MOLECULAR CELL BIOLOGY

WHAT IS GENE THERAPY ???Definition: an experimental technique for correcting defective genes that are responsible for disease development.

TYPE 1 : Ex vivo

cells are modified outside the body and

then transplanted back in again

called ex vivo because the cells are treated

outside the body

TYPE 2 : In vivo

genes are changed in cells when the cells are

still in the body

called in vivo because the gene is transferred

to cells inside the patient’s body

HISTORY OF GENE THERAPY1960s – 1970s – growing debates on social and ethical implications accompanied .......... 1966 - first idea related about gene therapy was mentioned by Edward Tatum , he also coined the term “human genetic engineering”.1968- Lederberg mentioned the term “virogenic therapy’’His idea viruses could be used to transfer DNA molecules that could encode for a therapeutic entity into cells of patients suffering from hereditary defects .1969- the first isolation of a gene succeeded by Beckwith .Late 1960s – Stanfield Rogers failed protocol - Shope Papilloma Virus patient’s body (- arginase activity) Assumption – Virus contained arginase gene induce arginase expression preferential growth of cells with higher arginase activity ........ Result - failed !!!! No arginase effect in patients body !!! 1980 - cline and colleagues .. Again failed .. Protocol – β – globin gene human bone marrow cell thalassemia patient's body failed !!! BOTH TRAILS LACKED

SOUND PRACTICE WELL-PROVEN CELL CULTURE

HISTORY OF GENE THERAPY

1990 - The first gene therapy journal published, Human Gene Therapy1990 - The first approved gene therapy clinical trial took place when Ashanthi DeSilva, a 4 year old girl with ADA-deficient Severe Combined Immunodeficiency, was given her own T cells engineered with a retroviral vector carrying a normal ADA gene 2000 - The first gene therapy cure was reported when Alain Fischer (Paris) succeeded in totally correcting children with SCID-X1, or “bubble boy” syndrome

BONE •BONE - Bone is the main supporting system in the human body. It is a unique combination of minerals and tissue that provides excellent tensile and loading strength. Bone has an intrinsic healing capacity that may be exceeded when the fracture gap is toobig or unstable

• •What are bone grafts? Bone grafts are the materials used for replacement or augmentation of the bone. •Types of Bone Grafts : on the basis of source Autografts Allografts Xenografts

source is the patient , usually

from tibia , fibula or ilium.

Also rib.

source is an individual other than the patient.

derived from different species …..

I AM STRONG, YAHHHH!

BUT !!! OH NO …

Trauma , bone tumour res ections / arthritis lead to

larger bone defects COMPROMISED HEALING

VIRAL GENE TRANSDUCTION MOST used virus type : ADENOVIRUSES ADENO-associated viruses Lentivirus Retrovirus

Parker et al, 2003 adeno/retro virus vectors BMP encoding plasmid ‘’GAM’’ = gene activated matrix .. Used in in vivo for bone healing .. Based on loading of BMPs plasmid / vectors into BIOMETERIALS

implant at defect siteRAT FEMORAL DEFECTS : ADENOVIRAL constructs encoding BMP2 , Runx2 , VEGF . BMP2 MSC TRANSDUCED WITH runx2 VEGF ADENOVIRAL BMP2

Other Studies (besides rat) Rabbit , sheep, pig, goat rabbit femur segmental defect injection of BMP2 encoding adenovirus vector HEALED

Also in sheep treatment is successful .....

Recombinant viral vectors Widely used ability to infect cell

types with high efficiency ..

COLLAGEN , CHITOSAN ,POLYES

TERS etc..

Direct percutaneous

injection

healed femoral defects

Efficient healing

(Lieberman et al, 1999)

Induced higher bone

mineral density

Induced angiogenesis

able to promote bone formation

NON VIRAL GENE THERAPY Carriers : * Cationic polymers * Cationic liposomes WORK : # Adipose tissue-derived MSCs with a G4 PAMAM/BMP-2 plasmid dendriplex inducing this cells to differentiate into the osteogenic phenotype . # BMP-2 cDNA in an alginate hydrogel promising result #Hydrogel : Fibrin , hyaluronic acid deliver oestrogenic genes induced bone formation fracture healing

#SONOPORATION : ( ’’cellular sonication ‘’ ) , ultrasonic frequencies for modifying the permeability of the cell plasma membrane. .

BMP-2 , BMP-7 CO -EXPRESSION PLASMIDS

WIDELY UTILIZED CARRIERS

Opposite surface charge forming lipoplexes/polyplexes with –vely

charged Dna

SONOPORATION Transduction efficiency

+ INCREASED

- COMPARATIVELY decreased

luciferase plasmid and bioluminescence

OTHER THAN GROWTH FACTORS miRNA – microRNA ,  small non-coding RNA molecules. RNA silencing and post-transcriptional regulation of gene expression . miRNA 218 – PRO-OSTEOBLASTIC FACTORS acts on Wnt INHIBITION .. miRNA – 148a – PRO-OSTEOCLASTIC FACTOR BLOCKING MAFB SIGNALING ..

OTHER miRNAS miRNA 23a, 30c , 34c, 133a, 135a, 137, 204, 205, 217, 338 . STUDY :

Control Osteoblastic

differentiation

miRNA26a in hydroxalapatite –

tricalcium phosphate scaffolds

miRNA WITH MAGNETIC

NANOPARTICLES

miRNA-31 IN Polyglycol sebacate

TRANSFECTED MSC

INDUCED BONE FORMATION

SUBCUTANEOUSLY IMPROVED RESULT

IMPROVED THE HEALING OF A RAT

CRITICAL CALVARIAL DEFECT

OPTIMIZATION , EXPRESSION AND FUNCTION

Genes need to be locally expressed in specific target cells achieved by Using aptamers that specifically bind osteogenic progenitor cells .Level of gene expression needs to be controlled achieved by using molecular sensors and negative feedback loops . Period of expression needs to be controlled achieved by using TET-on / TET-off systems . Angiogenesis important for bone regeneration . achieved by BMPs and VEGF

Tetracycline-Controlled Transcriptional Activation is a method of inducible gene expression where transcription is

reversibly turned on or off in the presence of the antibiotictetracycline or one of its derivatives

(e.g. doxycycline)

CONCLUSION

•The most promising Combinations of different genes in association with biomaterials •clinical trial November 11th 2014 a GAM based oncollagen-hydroxyapatite including the gene for VEGF -A165 to treat alveolar bone loss •No other clinical trials are ongoing at the moment.• PROPOSITIONː gene therapy for bone regeneration is still far away from clinical implementation but not out of reach .

ALL CREDITS GOES TO

DR. ENA RAY BANERJEE PROFESSOR DEPARTMENT OF ZOOLOGY UNIVERSITY OF CALCUTTA

..and special thanks to all my fellow classmates

THANK YOU.

THANK you for your KIND attention………….