gene therapy and its types

1. GENE THERAPY

2. GENE THERAPY
• Gene therapy is a strategy used to treat disease by correcting defective genes or modifying how
genes are expressed.
• The techniques used involve administrating a specific DNA or RNA sequence.
• The first approved gene therapy experiment occurred on September 14, 1990 in US, when Ashanti
DeSilva was treated for ADA-SCID.
• Gene therapy will enable patients to be treated by inserting genes into their cells rather than
administering drugs or subjecting them to surgery.
• This therapy offers a promising new approach to treating a range of diseases including various
forms of cancer, inherited disease and certain viral infections.

4. OUTCOME
• Replaces a mutated gene with a healthy one.
• Deactivates a gene that isn’t functioning properly.
• Introduces a new gene in the body to help fight the disease.
• Enhances the effect of a normally functioning gene.
• Activates the gene that was shut down during fetal life.

5. TWO MAIN APPROACHES IN GENE THERAPY:
1. INVIVO GENE THERAPY
2. EXVIVO GENE THERAPY
TWO TYPES OF GENE THERAPY BASED ON THE
CELLS
1. GERMLINE GENE THERAPY
2. SOMATIC CELL GENE THERAPY

6. INVIVO GENE THERAPY
• Delivery of corrected copy of the gene systemically through injection is a highly efficient
way to transfer a transgene to the patient's body.
• The major problem of in vivo method is its inefficient targeting.
• The transgene delivered into the body by means of viral or non viral vector also evokes
the immune response.
• The immune response against the vector leads to its clearance and only transient
expression of transgene.
• The neutralizing antibody does not allow the second injection of the vector.

7. • All gene therapy delivery protocols require the transgene to cross the
plasma membrane and enter inside the nucleus.
• The major obstacle is still to deliver the transgene effectively to the
intracellular compartment.
• Many modifications have been suggested into the viral vectors and also
non viral vectors to target the gene to the tissue.
• Examples: Fibroblast cells, Hematopoietic cells.

9. Advantages
• Simplicity.
• Gene delivery is accomplished by the single step of direct vector injection into the desired
target organ to correct the disorders.
• This technique has minimal invasiveness.
• This is due to the procedures involved in this technique is simple and safe.
• The same location can be injected more than once using in vivo gene delivery approaches.
Disadvantages
• The non specificity of target cell infection.
• Various different cell types can be infected when in vivo vectors are injected in the CNS,
including neurons, glia, and vascular cells.
• Besides, this technique might causes toxicity.
• Some in vivo vectors are toxic to host cells and elicit immune responses.

10. EXVIVO GENE THERAPY
• A technique where genetic manipulation of cells is undertaken remotely and more
safely since it is outside the body.
• It is an emerging therapeutic strategy particularly well suited to target a specific organ
rather than for treating a whole organism.
• The eye and visual pathways therefore make an attractive target for this approach.
• With blindness still so prevalent worldwide, new approaches to treatment would also
be widely applicable and a significant advance in improving quality of life.

11. • Ex vivo gene therapy has already achieved significant advances in the
treatment of blindness in pre-clinical trials.
• In particular, advances are being achieved in corneal disease, glaucoma,
retinal degeneration, stroke and multiple sclerosis through genetic re-
programming of cells to replace degenerate cells and through more
refined neuro-protection, modulation of inflammation and replacement
of deficient protein.
• Examples: Fibroblast cells, Hematopoietic cells.

13. Advantages
• Its ability to target selectively specific cell types for production of the gene of interest before
engrafting of cells into the host.
• It is immuno-compatible because the cells are collected from patient to avoid rejection.
Disadvantages
• It has to be maintained and genetically modified in vitro, host cells must be capable of
dividing, thus certain post mitotic cell populations such as neurons cannot be targets of
transduction for ex vivo gene therapy.
• It is also invasive.
• Grafting of cells is an intrinsically more invasive process than injection of suspensions of in
vivo gene therapy vectors.

15. GERM LINE GENE THERAPY
• Permanent transfer of the gene to the sperm or egg cells.
• Permanent changes that are passed down to subsequent generations.
• Not permitted in any country, on the basis that it is unethical.
• Theoretically, this approach should be highly effective in
counteracting genetic disease and hereditary disorders, but at present,
a variety of technical difficulties and ethical reasons make it unlikely
that germ line therapy would be tried in human beings in near future.

17. Advantages:
• It offers the possibility for a true cure of several diseases and it is not
only a temporary solution.
• It might be the only way to treat some genetic diseases.
• The benefits would be extended for several generations, because genetic
defects are eliminated in the individual’s genome and, consequently, the
benefits would be passed to his or her offspring.

18. Disadvantages:
• It involves many steps that are poorly understood, and the long-term results
cannot be estimated.
• It would open the door for genetic modifications in human traits with profound
social and ethical implications.
• It is very expensive and it would not benefit the common citizen.
• The extension of the cure to a person’s offspring would be possible only if the
defective gene was directly modified, but probably not if a new gene was
added to another part of the genome.

19. SOMATIC CELL GENE THERAPY
• Somatic gene therapy only involves the insertion of therapeutic DNA into body
cells and not the germ cells or gametes. This means any effects of the therapy are
confined to the individual being treated and are not inherited by future offspring.
• Single defective cell taken out of an individual’s body.
• Functional version of gene introduced into cell in a laboratory.
• Cells reproduce.
• Copies of cells with a corrected version of the gene is injected back into the
patient the good gene ends with the patient and is not inherited by their offspring.

21. Advantages:
• It is relatively effective as a procedure.
• It is less controversial then germ line gene therapies.
Disadvantages:
• If a treatment is successful, it will not be passed on to the patient/organism’s
offspring.
• Use of viral vectors is difficult.

22. GENE TRANSFER METHODS
• To transfer the desired gene into a target cell, a carrier is required. Such
vehicles of gene delivery are known as vectors.
• Vectors are needed since the genetic material has to be transferred across the
cell membrane and preferably in to the cell nucleus.
• Two main classes
1. Viral vectors
2. Non viral vectors

23. VIRAL VECTORS
• Viruses introduce their genetic material into the host cell as part of their
replication cycle.
• By removing the viral DNA and using the virus as a vehicle to deliver
the therapeutic DNA.
• The viruses used are altered to make them safe, although some risks
still exist with gene therapy.

24. ADENO VIRUS VECTOR
• Adenoviruses are large linear double-stranded DNA viruses that are commonly used for
preparing gene transfer vectors.
• Adenovirus vectors are known to be the second most popular gene delivery vector for gene
therapy of various diseases like cystic fibrosis and certain types of cancer.
• The adenoviruses enter cells by receptor-mediated endocytosis.
• A primary cellular receptor binds to viral fibre then the virus interacts with secondary
receptors which are responsible for its internalization.

25. • Coxsackie Adenovirus Receptor (CAR), Heparan sulphate glycosaminoglycans, Sialic acid,
CD46, CD80, CD86, alpha domain of MHC I are the primary receptors which are specific
for specific strains of Adenovirus.
• Integrins are the secondary receptors which helps in the internalization of viral particles.
• Some adenovirus directly interacts with integrins like in the case of fibre deficient Ad2
virions.
• The adenoviral DNA has inverted terminal repeats (ITRs) and a terminal protein (TP) is
attached covalently to 5’ termini.
• The adenoviral genome is classified as early and late regions based on the proteins they
express.

26. • Proteins encoded by early region (E1, E2, E3, E4) genes are involved in viral DNA replication, cell cycle
modulation and defence system.
• The late region genes (L1, L2, L3, L4, L5) encodes the viral structural proteins.
• Three classes of adenoviral vectors namely first, second and third generation viral vectors are developed for
gene therapy purpose.
First generation adenoviral Vectors
• These vectors are constructed by replacing the E1/E3 expression cassette and inserting gene of intrest of 3-
4kb size.
• E1 encodes proteins responsible for expressions of other viral genes required for viral growth.
• So cell lines that can provide E1 proteins in trans are required for the replication of the E1 deleted viral
vectors.

27. Advantages:
• They are human viruses produced at very high titters in culture. They can infect a wide range of
human cell types including non- dividing cells.
• They enter into cells by receptor mediated endocytosis with a very high transduction efficiency
reaching up to 100% in vitro.
• Their large size enables them to accept large inserts.
Disadvantages:
• Expression of foreign gene is for short period of time as they do not integrate into the chromosome.
• These vectors may generate immune response causing chronic inflammation.

28. Second generation adenoviral Vectors
• These vectors have been developed to overcome these difficulties.
• Here E1/E2 or E3/E4 expression cassettes are deleted and replaced.
• The E1/E2 or E3/E4 proteins are required for viral DNA replication.
• Similar to first generation viral vector, cell lines which can complement both E1and E2 or E3 and E4 are needed.
• It can carry DNA insert upto 10.5kb.
Advantages:
• It has improved safety and increased transgene expression.
Disadvantages:
• These viral vectors are associated with immunological problems.
• Construction of these vectors is difficult.

29. Third generation adenoviral Vectors
• These vectors are otherwise called as gutless adenovirus.
• These are also known as helper dependent adenovirus as they lack all the coding
sequences and require helper virus which carries all the coding sequences.
• Helper virus for example AAV, or artificially disabled viruses provide the viral
functions needed for successful infection like viral DNA replication, viral assembly
and infection of new cells etc.
• The size of insert DNA can be 36kb and hence called as high capacity adenoviruses.
• They carry only 5’ inverted terminal repeats (ITR) and 3’ packaging signals (ψ).

31. Advantages:
• These are non-integrative and high-capacity vectors.
• It can be produced in high titer and the construction of these vector is easy.
• It shows longer stability and reduced immune response.
Disadvantages:
• Helper virus contamination can cause diseases like conjunctivitis, pharyngitis,
cold and respiratory disease.

32. RETRO VIRUS VECTOR
• First viruses to be used as vectors in gene therapy experiments were retroviruses.
• They belong to a class of viruses which can create double stranded DNA copies with
the enzyme reverse transcriptase.
• These copies of its genome can be integrated into the chromosome of host cell by
another enzyme carried the virus called integrase.
• Now the host cell has been modified to contain a new gene. If such modified host
cells divide later, their descendants will contain the new genes.

33. • Gene therapy trial using retroviral vector to treat x-linked severe
combined immune deficiency represent the most successful application
till date. Also this has been tried to treat SCID due to ADA deficiency
with relative success.

34. Advantages
• The fact that they are well known and well characterized, the possibility to well characterize them and to
produce them in high titters and their high transfection efficiency which has been validated in various cell
types.
Disadvantages
• An accommodation limit of 7-8 kb of DNA, the need that the target cells be in cell division to allow for the
integration of the vector, and the potential of insertional mutagenesis due to the fact that retroviral vectors
integrate at random in the genome.
• The latter feature has the potential to interrupt important genes in the cell, with serious consequences that
include oncogenesis through the activation of proto-oncogenes or inactivation of tumor suppressor genes.
• They limit their in vivo use include their inactivation by complement in serum, and the poorly understood
spontaneous inactivation of the LTR promoter function.

35. ADENO ASSOCIATED VIRUS VECTOR
• Adeno-associated viruses (AAVs) are a group of small, single-stranded
DNA viruses which cannot usually undergo productive infection without
co-infection by a helper virus, such as an adenovirus.
• The insert size for AAV is 4.5 kb, with the advantage of long-term gene
expression as they integrate into chromosomal DNA.
• AAVs are highly safe as the recombinant adeno associated vectors
contains only gene of interest and 96% viral genes are deleted.

37. Advantages:
• Non pathogenic
• Broad host and cell type range.
• Transduce both dividing and non-dividing cells.
• Maintain high levels of gene expression over a long period of time in vivo.
Disadvantage:
• Smaller size limits the amount of foreign genes that can be inserted.
• Slow onset of gene expression.

38. Advantage of viral vectors
• Viral vectors have been employed for the treatment of various diseases such as metabolic,
cardiovascular, muscular, hematologic, ophthalmologic, and infectious diseases and
different types of cancer.
• Recent development in the area of immunotherapy has provided both preventive and
therapeutic approaches.
• Gene silencing generating a reversible effect has become an interesting alternative, and is
well-suited for delivery by viral vectors.
• A number of preclinical studies have demonstrated therapeutic and prophylactic efficacy in
animal models and furthermore in clinical trials.
• Several viral vector-based drugs have also been globally approved.

39. NON VIRAL VECTOR
It involves chemical and physical methods such as direct injection
of naked plasmid DNA (particle bombardment), receptor-mediated
endocytosis and gene transfer through liposomes, polymers, nano-
particles etc. are some non viral methods.
1. Physical method.
2. Chemical method.

41. ELECTROPORATION
• In electroporation, the external electric field is applied to the protoplast,
which changes the electrical conductivity and the permeability of cell
membrane; and thus the exogenous molecules found in the medium are
taken up to either the cytoplasm or into the nucleus.
• The efficiency of electroporation can be increased by giving the cell a
heat shock, prior to the application of electric field or by using small
quantity of PEG while doing electroporation.

43. Advantage:
• By electroporation large numbers of cells can be processed at once, and thus
the amount of time spent processing cells can be cut down.
Disadvantages:
• If the voltage applied is not calculated properly, the cells may damage.
• If electroporation does not occur in controlled environment, the potentially
harmful substances can enter the cell or the impurities from solution may
enter.
• This is because there is no way to control what enters the cell membrane.

44. MICROINJECTION
• Microinjection involves the delivery of foreign DNA, by the help of
glass micropipette into a living cell.
• The cell is held against a solid support or holding pipette and micro
needle containing the desired DNA is inserted into the cell.
• The tip of the pipette used is about 0.5 to 5 micro meter diameter
which resembles an injection needle.

45. • For this, glass micropipette is heated until the glass becomes somewhat
liquefied and is quickly stretched to resemble a injection needle.
• The delivery of foreign DNA is done under a powerful microscope.

46. Advantages:
• Frequency of suitable integration of DNA is far better as compared to other methods.
• Method is effective in transforming primary cells as well as cells in established cultures.
• The DNA injected in this process is subjected to less extensive modifications.
• Mere precise integration of recombinant gene in limited copy number can be obtained.
Disadvantages:
• Costly.
• Skilled personal required.
• More useful for animal cells.
• Embryonic cells preferred for manipulation.
• Knowledge of mating timing, oocyte recovery is essential.
• Method is useful for protoplasts and not for the walled cells.

47. GENE GUN METHOD
• DNA can be injected parentally which can be considered for Duchenne muscular
dystrophy (DMD).
• An alternative approach uses particle bombardment technique, in which DNA is coated
on to metal micro particles and fired from a ballistic gun into cells/tissues.
• This technique is used to transfer the foreign DNA and its transient expression in
mammalian cells in vitro and in vivo as well.
• It can cross the physical barriers like skin, muscle layer for which it is used for
vaccination.
• Particle bombardment is used to deliver drugs, fluorescent dyes, antigenic proteins etc.

49. Advantage:
• Simple and comparatively safe.
Disadvantage:
• Poor efficiency of gene transfer.
• A low level of stable integration of the injected DNA.
• Repeated injection may cause damage in the proliferating cells.

51. LIPOSOMES
• Liposomes are spherical vesicles which are
made up of synthetic lipid bilayers which imitate
the structure of biological membranes.
• DNA to be transferred is packaged into
the liposome in vitro and transferred to to the targeted tissue.
• The lipid coating helps the DNA to survives in vivo and
enters into the cell by endocytosis.
• Cationic liposomes, where the positive charge on
liposomes is stabilized by binding of negatively charged DNA,
are popular vehicles for gene transfer in vivo.

52. Advantage:
• The liposomes with the foreign DNA are easy to prepare.
• There is no restriction in the size of DNA that is to be transferred.
Disadvantage:
• Efficiency of gene transfer is low and transient expression of the foreign
gene is obtained as they are not designed to integrate into the
chromosomal DNA.

53. RECEPTOR MEDIATED ENDOCYTOSIS
• It can be both viral and non-viral mediated gene transfer.
• Viral vectors attach to the surface receptors through viral surface components and internalized.
• In non viral mode of receptor mediated endocytosis DNA is first coupled to a ligand that binds
specifically to a cell surface receptor and causes transfer of the DNA into cells by endocytosis.
• Coupling is done by linking the receptor molecule with poly lysine followed by reversible binding
of the negatively charged DNA to the positively charged poly lysine component.
• Transferrin receptor which is comparatively abundant in proliferating cells and hematopoietic cells
is utilized as a target and transferrin as a ligand in this approach.

55. Advantage:
• Gene transfer efficiency may be high.
Disadvantage:
• It does not allow integration of the transferred genes. Also, the protein—
DNA complexes are not stable in serum.
• Coupling of inactivated adenovirus to the DNA-transferin complex can
increase gene transfer efficiency which help in receptor mediated
endocytosis and lysosomal escape.

56. ADVANTAGES OF NON – VIRAL VECTORS
• Due to its demonstrated reduced pathogenicity, low cost and ease of
production, non-viral vectors have important safety advantage over viral
approaches.
• The major advantage of using non-viral vectors is its bio-safety.
• its less immunotoxicity.

57. ADVANTAGES OF GENE THERAPY
• Gene therapy can cure genetic diseases by addition of gene or by
removal of gene or by replacing a mutated gene with corrected gene.
• Gene therapy can be used for cancer treatment to kill the cancerous
cells.
• Gene expression can be controlled.
• Therapeutic protein is continuously produced inside the body which
also reduces the cost of treatment in long term.

58. DISADVANTAGES OF GENE THERAPY:
• Due to the fact that this is still a new scientific breakthrough, the safety concerns outweigh quite a bit of the
procedures. The current understanding of gene therapy is based on theory rather than fact.
• Irregular immune responses.
• Viral vectors may introduce toxicity, as well as immune and inflammatory responses.
• Multi-gene disorders such as heart disease, high blood pressure, Alzheimer’s disease, arthritis, and diabetes cannot
be treated through this therapy as conditions or disorders that arise only from mutations in a single gene are the best
candidates for gene therapy.
• Religious concerns.
• Chances of inducing iatrogenic tumours in human beings.
• Short-lived nature of gene therapy.

59. CONCLUSION
 Theoretically, gene therapy is the permanent solution for genetic diseases.
 But it has several complexities. At its current stage, it is not accessible to
most people due to its huge cost.
 A breakthrough may come anytime and a day may come when almost every
disease will have a gene therapy
 Gene therapy have the potential to revolutionize the practice of medicine.

60. Reference:
• Ben-Israel, H., And Kleinberger, T. (2002). Adenovirus And Cell Cycle Control. Front. Biosci. 7, Dl369–D1395.
• Nayerossadat, Et Al.: Viral And Nonviral Delivery Systems For Gene Delivery, Advanced Biomedical Research, Vol 1 Issue 2.
• Najmul Hasan Et Al., Gene Therapy: Current Status And Future Perspectives, International Journal Of Pharma Sciences And Research, Vol 5 No 09 Sep 2014
• Torsten O. Nielsen, Human Germline Gene Therapy, Mjm 1997 3: 126-132
• Torsten O. Nielsen, Human Germline Gene Therapy, Mjm 1997 3: 126-132
• Y. Touchefeu Et.Al., Review Article: Gene Therapy, Recent Developments And Future Prospects In Gastrointestinal Oncology, Aliment Pharmacol Ther
2010; 32: 953–968
• E. N.; Kostyk, S. K.; Thomas, K. Et Al. (2011). "Aav2-Gad Gene Therapy For Advanced Parkinson's Disease: A Double-Blind, Sham-Surgery Controlled,
Randomised Trial". The Lancet Neurology 10 (4): 309–319.
• Masaki Uchida, Xiong Wei Li, Peter Mertens, H. Oya Alpar (2009). Transfection By Particle Bombardment: Delivery Of Plasmid Dna Into Mammalian Cells
Using Gene Gun. Biochimica Et Biophysica Acta 1790, 754–764
• Ana P. Cotrim And Bruce J. Baum, Gene Therapy: Some History, Applications, Problems, And Prospects, Toxicologic Pathology, 36:97-103, 2008.
• Anne Cathrine Staff , An Introduction To Gene Therapy And Its Potential Prenatal Use ,Acta Obstet Gynecol Scand 2001: 80: 485–491