This document discusses the role of genetics in orthopaedics. Gene therapy can be used to treat conditions like impaired bone healing, cartilage repair, and metabolic bone diseases by inserting genes to modulate gene expression in cells and tissues. There are two main methods of gene delivery - in vivo, where genes are directly transferred to the host, and ex vivo, where genes are transferred to cell cultures before being reintroduced to the subject. Common gene therapy applications in orthopaedics include treating rheumatoid arthritis, osteoarthritis, bone healing and repair, cartilage repair, and osteoporosis. Overall, gene therapy has proven feasibility but more research is still needed before it can be widely used in clinical orthopaedics.

1. ROLE OF GENETICS IN
ORTHOPAEDICS
PRESENTED By: Dr. B.Borthakur
Professor
Dept of Orthopaedics, SMCH

2. INTRODUCTION
• Orthopaedic disease can cause changes in biological
signalling at the tissue level that potentially can be repaired
or modified by inserting genes into the cells or tissues to
modulate gene expression.
• Impaired bone healing, need for extensive bone formation,
cartilage repair and metabolic bone diseases are all
conditions where alterations of the biological signalling may
provide cure or improvement.

3. PRINCIPLES
• Gene therapy nowadays exist to modify genetic structure or
gene encoding.
• Treatment is designed by inserting genes into an individual’s
cells and tissues or replacement of mutant gene in hereditary
diseases.

4. • In general, gene delivery is performed in two ways.
• In vivo – where gene is transferred directly to the host.
• Ex vivo – where gene is transferred outside the body to a
cell or tissue cultures then culture reintroduced into living
subject

5. For orthopaedics application, before gene transfer, the genes of interest
are typically encoded for peptide growth factors that are able to enhance
or initiate bone formation, repair cartilage or other tissues or induce
programmed cell death (apoptosis) when required in tumour tissues

6. • In ex-vivo gene therapy, the modified cells besides delivery
genes also functions as a drug delivery system providing more
increased and prolonged local extra-expression of the peptide
growth factors.

8. • The transferred gene is called Transgene and the
organism/tissues that develop after a successful
gene transfer is known as Transgenic.
• The vehicles that are responsible for gene
delivery are called Vectors.

9. DELIVERY SYSTEMS OF GENE
• VIRAL DELIVERY SYSTEM
• NON-VIRAL DELIVERY SYSTEM
• VIRAL DELIVERY SYSTEMS:
• Viruses are nature’s own gene therapy organism with their
ability to invade cells and deliver genetic material to the nucleus
of the organism/tissue for incorporation into the genome.
• 1.Retroviruses were the earliest vectors for gene delivery
• 2.Adenoviruses

10. NON-VIRAL DELIVERY SYSTEM:
• These systems do not actively apply biological system to
incorporate the desired DNA into the genome like viruses.
• The techniques used can be physical,mechanical and chemical.

11. PHYSICAL METHODS
• DNA injection directly into cells eg.smooth muscle
• Electroporation
• Lipofection in where DNA complexed with lipid vesicles
are taken up by cells via endocytosis

12. ELECTROPORATION
• is a method where electrical energy causes formation of
pores increases cell permeability to facilitate DNA
molecules flux into the cell.
• It has been found easy to perform
• High efficiency
• Do not alter cell biology

14. LIPOFECTION
Technique used to inject genetic materials into the
tissues by means of liposomes.
Liposomes are artificial phospholipids vesicles to deliver
gene to the target tissues

16. CHEMICAL METHODS, it uses synthetic vectors
• DNA transfer by Calcium phosphate method
• DAEA-Dextran for DNA transfer
• Liposomes mediated transfer

17. DNA TRANSFER

18. MECHANICAL METHOD
GENE GUN
• DNA is coated with gold particles and loaded into a device which
looks like a gun and is injected into the target cell/tissues.

19. APPLICATIONS OF GENE THERAPY

20. RHEUMATOID ARTHRITIS
• First human application in arthritic Gene therapy
• Ex-vivo delivery system
• VECTOR:Retrovirus to deliver GENE Interleukin-1 receptor
antagonist (IL-1 Ra)
• Evans et al. used this therapy in metacarpophalangeal joints
that were about to undergo sialistic joint replacement surgery
or synovectomy
• Studies showed procedure was safe,feasible,marked clinical
improvement in human

21. OSTEOARTHRITIS

22. IN-VIVO
• Local gene transfer
• Direct Intra-articular injection of Hyaluronan
synthase 2 GENE into the individual joint disease

23. EX-VIVO
• Cells cultured with required gene is injected and adhere
to the joint line to deliver the gene product to target
tissues
• VECTOR: Adeno-associated Virus
• GENES: IL-1 Receptor antagonist
Insulin growth factor-1
GFAT gene (glutamine-fructose-6-phosphate
transaminase 2)
• Increase the joint space tissues to synthesize
glucosamine

26. BONE HEALING AND REPAIR
• Impaired or inadequate bone repair such as fracture
non-union, implant loosening, large bone defects after
trauma /tumour resections.
• In these conditions, genes encoded for osteoinductive
factors are used
IN-VIVO GENE THERAPY
• Vector - Adenovirus
• Gene - Osteoinductive factor- BMP-2 growth factor
• Injected directly into the target tissue/intraarticular

27. EX-VIVO GENE THERAPY
• Non-viral methods
• Aim is osteogenic differentiation
CELL BASED GENE THERAPY
• MSCs and primary marrow drived stem cells
• GENE: BMP-2 (bone morphogeneticprotien-2)
COLLAGEN-based gene activated matrix
• GENE: BMP-4 and PTH 1-34.
• Ex-vivo gene therapy offer the advantage of selecting cells for delivery
• Also,no viral particles/DNA are inserted into the patient

29. WEAR DEBRIS-INDUCED OSTEOLYSIS
• Gene transfer via AdenoAssociatedVirus vector has shown
protective effects against orthopaedic wear debris-induced
bone loss

30. ASEPTIC LOOSENING
• De pooerter et al. adopted a gene transfer approach
Recombinant Adenovirus (VECTOR)
Nitrate reductase gene was injected
Intraarticularly.
Prodrug CB 1954
Nitrate reductase gene coverted this prodrug to a cytotoxic product within the
synovium.
Target cells killed
Liquid bone cement introduced into the space to restabilize the prosthesis
• Test subjects showed reduced pain and increased walking distance

31. SPINAL FUSION
• In spinal fusion surgery,bone graft is used to form a bridge between
two vertebral segments in the spine to form fusion.
• Failure of solid fusion is a significant problem.
• Gene therapy using Non-viral delivery sysstem
• Genes encoding for osteoinductive protiens within the BMP family
could be potentially used
for cell-based gene therapy.
• LIM MINERALIZATION PROTIEN-1 (LMP-1) gene has been successfully
used
• It regulates expression of numerous growth factors in the BMP family
showing increased biological response.

32. CARTILAGE REPAIR
• Cartilage defects, generalised degenerative lesions
• Non-viral
• Cell-based gene therapy
• Growth factor genes used are-
Insulin-like growth factor (IGF)
Fibroblast growth factor 2
Transforming growth factor-beta(TGF-beta)
Bone morphogenetic protiens (BMP)
• Gene transfer into articular cartilage and synovial lining cells
• This gene therapy focuses on cartilage matrix homeostasis and
chondrogenesis

34. LIGAMENT HEALING
• With gene therapy
• Use of BMP-2 or BMP-7 in an injectable calcium phosphate
matrix in rabbit ACL improved tendon to bone healing on
histologic and biochemical testing

35. TENDON HEALING
• Adenovirus with BMP-14 AND BMP-12 INCREASED TENDON
TENSILE STRENGTH IN A RAT ACHILLES TENDON INJURY
• Increase in expression of type 1 and type 2 collagen mRNA
helps repair of tendons and ligaments

37. DEGENERATIVE DISC DISEASE
• Chronic condition of the disc tissue degeneration leading to
loads resistance
• Loss of proteogylcans and water in the disc tissue.
• Gene therapy aims to restore proteoglycans and water in
the disc tissue
• Growth factors are used
TGF-beta
IGF
BMP
PDGF

38. • Nucleus pulposus cellular longevity by Telomerase gene
therapy
• Telomerase can extend the cellular lifespan of nucleus
pulposus cells.

39. OSTEOPOROSIS
• Bone loss and osteopenia
• Type 1 osteoporosis- increased osteoclastogenesis due to oestrogen depletion
• Type 2 osteoporosis- decreased osteogenesis due to senescence of marrow stem
cells.
• Non-viral
Type 1 osteoporosis
• The aim is to Decrease osteoclastogenesis by inhibiting RANKL
• RANKL (receptor activator of NF-kappa beta ligand) is responsible for
osteoclastogenesis
• OPG (osteoprotegerin) can inhibit the RANKL
• Marrow stem cells transfected with the gene for OPG and reintroduced into the
osteoporotic tissue could result in systemic inhibition of osteoclastogeneis.

40. • Type 2 osteoporosis
• Marrow stem cells from osteoporotic donors are transduced with
adenovirus vectors carrying the BMP-2 gene

42. OSTEOPETROSIS
• Excessive bone formation resulting in marrow obliteration
• Excessive bone formation is due to osteoclastogenesis
• Genetic defect in gene coding CSF-1 ,Colony stimulating factor-1
• Gene therapy- incorporate marrow stem cells that over-expresses CSF-
1 gene leading to increased osteoclastogenesis.

43. HETEROTOPIC OSSIFICATION
• Abnormal growth of bone in non skeletal tissues like
muscles,tendons or other soft tissues
• Responsible gene BMP
• Adenovirus mediated transfer of RNA against runx2/Cbfa 1
inhibits the formation of heterotopic ossification induced by
BMP4 (in animal model)

44. OSTEOGENESIS IMPERFECTA
• Genetic disease causing reduced bone strength and susceptibility to
fractures
• Due to mutation in pro-collagen 2 (I) chain
• Gene therapy-based delivery of the pro-collagen could corect the
biochemical disorder

46. ONCOLOGY
• Primary bone tumours and bone metastases
• Aim is to accelerate tumour-tissue necrosis by inserting genes that
induce programme cell death.
• Gene TRAIL ,TNF-related apoptosis-induced ligand during exposure
with different chemotherapeutic agents was able to accelerate bone
tumour cell death.
• Gene therapy can also improve cancer treatment by introducing
genes that causes more cellular sensitivity to chemotherapy

49. RADIOTHERAPY
• Novel cytosine deaminase fusion gene enhances the effect of
radiation on bone tumours

50. CONCLUSIONS
• Gene therapy has proven its feasibility in orthopaedics
• Gene therapy offerrs to provide prolonged local drug delivery and with different cell
types can stimulate synergistic tissue responses
• Approaches to orthopaedic gene therapy rest on a solid conceptual and scientific
footing
• Pre-clinical, clinical datas
• There is therefore a considerable need for continuing research before regimens are
developed to a degree where we can use in clinical orthopaedics
• In orthopaedics,gene therapy will primarily be used for non-lethal conditions
because patient’s safety is of maximal importance
• An increased morbidity and mortality will be unacceptable in orthopaedics gene
therapy

51. • Chapter 2, Verse 22
“vaasaamsi jiirnaani yathaa vihaaya, navaani grihnaati naro
aparaani |
tathaa shariiraani vihaaya jiirnaanyanyaani samyaati navaani dehii
||”
Just as a person casts off worn out garments and puts on others
that are new, even so, the embodies soul casts off worn out bodies
and takes on others that are new.
THANK YOU..