Gene Therapy in Orthopaedics.pptx

What is Gene Therapy?
Gene therapy is an experimental technique that uses genes to treat or prevent disease

It focuses on the utilization of the therapeutic delivery of nucleic acids into a patient's cells as a drug to treat disease.

treatment directed to cure genetic diseases by introducing normal genes into patients to overcome the effect of defective genes. These genes deliver individual proteins to specific cells.
Gene therapy in Orthopaedics:
Potential applications of gene therapy as relevant to orthopedics:
Fracture healing
Non-union
Delayed-union
Compound fractures
Grade 3 open fractures.
- Ligament healing
Regeneration of ligaments like patellar tendon (extensor mechanism), ACL or improved fixation and incorporation of grafts
Meniscal tears
- Chondral (articular cartilage) healing and regeneration
- Muscular diseases
Muscular dystrophies
Muscle strain
Utilization of muscle cells as ex vivo delivery vehicles
Osteoarthritis (OA)
Primarily chondral regeneration
Alteration of joint milieu to retard joint degeneration
Rheumatoid arthritis
Altering or remitting the inflammatory process.
Types and Delivery systems of Gene Therapy --
Somatic gene therapy : targets non-reproductive cells

Germ line gene therapy : targets gamete cells – manipulations are passed to progeny

Viral vectors - done by viral transduction
genetically engineered virus permitted to initially infect the cell -- further replication inside cell is prevented
can not infect other cells d/t deficiency in replication
eg - ADV, AAV, HSV, Retrovirus

Non viral vectors - done by transfection (eukaryotic cells made to take up foreign DNA from environment)
- not as effective as viral vectors
- Naked DNA
- DNA -protein complex
- Liposomes (for delivering genes to chondrocytes)
- Gene gun (for muscles and cartilage cells)
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Systemic gene therapy
- targets all cells
- injected directly to blood
- treating metastatic diseases
- poor penetration in tissues with decreased blood supply – cartilage and menisci
Local gene therapy
- injected to specific tissue
Direct
- ACL, MCL, Meniscus, Tendon, Ligament
Indirect
- target cell is removed form body – exposed to vector in vitro and reinserted
- spine, articular cartilage, skeletal muscle

Gene delivery systems are categorized as viral-based,�non-viral-based and combined hybrid systems -
commonly used viral vectors consist of adenoviruses, retroviruses, and lentiviruses
newer viral vectors that have been developed to improve upon the existing delivery systems like:
- Sendai Virus Vectors (SeV) has been developed as a recombinant viral vector.

Physical methods: Microinjection, electroporation, ultrasound, gene gun, and hydrodynamic applications, etc.
Chemical methods : polymers, liposomes, dendrimers, and cationic lipid systems, etc.

References - Essential Orthopaedics – Principles and practice by Manish Kumar Varshney

1. Gene Therapy in Orthopaedics Dr. Vivek Jadawala PGY-2, Dept . of Orthopaedics JNMC, DMIHER

2. What is Gene Therapy? Gene therapy  is an experimental technique that uses genes to treat or prevent disease It focuses on the utilization of the therapeutic delivery of nucleic acids into a patient's cells as a drug to treat disease . treatment directed to cure genetic diseases by introducing normal genes into patients to overcome the effect of defective genes. These genes deliver individual proteins to specific cells

3. Gene therapy : Background

4. Gene therapy in Orthopaedics Fracture healing Non-union Delayed-union Compound fractures Grade 3 open fractures. - Ligament healing Regeneration of ligaments like patellar tendon (extensor mechanism), ACL or improved fixation and incorporation of grafts Meniscal tears P otential applications of gene therapy as relevant to orthopedics:

5. - Chondral (articular cartilage) healing and regeneration - Muscular diseases Muscular dystrophies Muscle strain Utilization of muscle cells as ex vivo delivery vehicles Osteoarthritis (OA) Primarily chondral regeneration Alteration of joint milieu to retard joint degeneration Rheumatoid arthritis Altering or remitting the inflammatory process.

6. Types and Delivery systems of Gene Therapy

7. Somatic gene therapy  : targets non-reproductive cells Germ line gene therapy  : targets gamete cells – manipulations are passed to progeny V iral vectors - done by viral transduction genetically engineered virus permitted to initially infect the cell -- further replication inside cell is prevented can not infect other cells d/t deficiency in replication eg - ADV , AAV, HSV, Retrovirus N on viral vectors - done by transfection (eukaryotic cells made to take up foreign DNA from environment) - not as effective as viral vectors - Naked DNA - DNA -protein complex - Liposomes (for delivering genes to chondrocytes) - Gene gun (for muscles and cartilage cells)

8. Systemic gene therapy - targets all cells - injected directly to blood - treating metastatic diseases - poor penetration in tissues with decreased blood supply – cartilage and menisci Local gene therapy - injected to specific tissue Direct - ACL , MCL, Meniscus, Tendon, Ligament Indirect - target cell is removed form body – exposed to vector in vitro and reinserted - spine , articular cartilage, skeletal muscle

9. Gene delivery systems are categorized as viral-based, non-viral-based and combined hybrid systems Viral-mediated gene delivery systems commonly used viral vectors consist of adenoviruses, retroviruses , and lentiviruses newer viral vectors that have been developed to improve upon the existing delivery systems like: - Sendai Virus Vectors ( SeV ) has been developed as a recombinant viral vector. Non-viral systems are divided into: Physical methods: Microinjection, electroporation, ultrasound , gene gun, and hydrodynamic applications , etc. Chemical methods : polymers , liposomes, dendrimers , and cationic lipid systems, etc.

10. Physical Methods Gene Gun (Ballistic DNA delivery or DNA-coated particle bombardment) Electroporation is controlled electric application -> increase cell permeability -> foreign genes into the cell by electric pulses.

11. Physical Methods Ultrasound - microbubbles applied by ultrasound increased gene expression. The microbubbles were modified with plasmid DNA before the injection Hydrodynamic Therapy - hydrodynamic pressure created by the injection of the large volume of DNA solution with blood pressure inside veins. The permeability of the capillary endothelium increases and pores form in the plasma membrane

12. Chemical Methods Polymers - A unique application of nanotechnology. Polymeric gene transporters deliver genetic material through electrostatic interactions with nano -sized polyplexes for gene therapy. This system of gene delivery is based on three strategies namely electrostatic interaction, encapsulation and adsorption Some polymeric vectors are as follows: Poly (L-lysine) (PLL)-based gene delivery systems Folate-conjugated PLL Sugar-conjugated PLL Antibody-PLL conjugates Arterial-wall binding peptide (AWBP)- conjugated PLL The terplex system. Polyethylenimine (PEI)-based gene carriers PEI-PEG Folate-conjugated PEIs PEI conjugates with targeting moieties.

13. Dendrimers – Dendrimers consist of star-shaped symmetrically branched macromolecules projecting from a central core (Fig. 4). They have highly controllable chemical behaviors and undergo perfect encapsulation capable of carrying material within. Cationic Lipid Compatible Systems ( Lipoplexes) – The complex of DNA with lipids is called lipoplex (Fig. 5 ). Lipoplexes electrostatically interact with the cell membrane and are then taken up.

14. Novel Non-viral Systems to Achieve Efficient Transgene Integrations Transposons are DNA sequences, which work by their enzymes named transposase, with help of which they can move from one chromosomal location to another . Hybrid Vectors:- Types : Virosomes—produced by the fusion of lipoplexes with inactivated HVJ virus (hemagglutinating virus of Japan) or influenza virus. Hybrids generated by mixing cationic liposomes or polymers with adenoviral vector. Hybrid viruses—produced by a combination of various types of viral vectors.

15. NEWER APPROACH: NANOPARTICLES AND NANOTECHNOLOGY Nanoparticles (size 5–200 nm) mainly maghemite ( c-Fe2O3) and magnetite (Fe3O4) nanoparticles  to improve the efficacy and the development of new therapies Others - Quantum Dots ( Qdots ) and Magnetic Nanoparticles

16. TARGETED GENE DELIVERY The most commonly used target  muscle tissue. Properties that make muscle tissue an appropriate target: Facilitation and long-term expression of introduced genes Myogenic precursor cells (satellite cells) can be relatively easily isolated and cultivated in vitro and efficiently transduced using either viral or non-viral vectors. The ability of the myogenic cells to fuse with or into myofibers . The high level of vascularization of muscle tissue facilitates the systemic delivery of potentially therapeutic proteins, such as growth factors, factor IX, or erythropoietin.

17. GENE THERAPY APPLICATIONS FOR THE MUSCULOSKELETAL SYSTEM Fracture healing and nonunion : the transfer of the genetic sequence or cDNA for one of these growth factors (i.e. bone morphogenetic protein) could be used to enhance fracture healing or treat non-unions . Growth factors with osteo - inductive properties are - bone morphogenetic proteins (BMP), - TGF-B , - PDGF , - IGF Growth factors with osteo -inductive and angiogenic both properties are FGF . LMP-1 is an osteogenic factor that is a part of the BMP pathway and is a critical regulator of osteoblast differentiation .

18. Osteoporosis – local growth factors or cytokines generated in the local bone cell microenvironment should have a significant influence on bone remodeling. The growth factors useful in treatment of osteoporosis are TGF-B, BMP, IGF, PDGP, and FGF. A potential method of the delivery of recombinant growth factors is to use genetically manipulated cells like bone marrow cells.

19. Cartilage defects – chondrocytes do not possess the inherent ability to regenerate themselves. Presently, treatment of cartilage defects includes autologous chondrocyte, drilling into subchrondral bone to facilitate migration of chondro -progenitor Growth factors known to enhance chondrogenesis are TGF-B and BMP-2. Autologus chondrocytes can be successfully harvested, grown in tissue culture with vectors containing genes for these growth factors, and reimplanted into an articular defect.

20. Osteoarthritis - Target cells in OA joint include chondrocytes, osteocytes, cells of the synovial lining , etc G ene transfer to chondrocytes – to compensate for mutations in structural proteins of the cartilage matrix to secrete chondroprotective agents. Human chondrocytes - transduced with an adenovirus containing the cDNA for interleukin receptor antagonist protein (IL-Ira ). IL-1ra is chondroprotective .

21. Other applications - Rheumatoid arthritis indirect gene therapy synovial cells harvested – transduced to produce IRAP – injected back to diseased joint IRAP blocks IL-1 intraarticularly

22. Other applications - Intervertebral disc direct delivery of TGF-beta to nucleus pulposus – increased proteoglycan synthesis

23. Other applications - Spine fusion - BMP-2 injections : solid fusion Local gene therapy injected to specific tissue direct ACL, MCL, Meniscus, Tendon, Ligament indirect target cell is removed form body – exposed to vector in vitro and reinserted spine, articular cartilage, skeletal muscle

24. In September 2010, it was announced that an 18-year-old male patient in France with beta-thalassemia major had been successfully treated. The technique used a lentiviral vector to transduce the human ß-globin gene into purified blood and  marrow cells obtained from the patient in June 2007 .  The patient's haemoglobin levels were stable at 9 to 10 g/ dL .

25. CONCLUSION Gene therapy is a newly emerging field which holds promise for future especially in management of genetic disorders . The success currently is limited in this technique to obtain complete cure however, introduction of advanced technologies and techniques emerging rapidly has obtained good response in animal models.

26. References Essential Orthopaedics – Principles and practice by Manish Kumar Varshney

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