Basic principles of genetics and the basic understanding of gene therapy and its applications in therapeutics.

1. Gene Therapy
Correcting defective genes
Dr. Mohit Kulmi

2. Basics of Genetics
• Nucleotides are the alphabets of DNA.
• Adenine (A), Thymine (T), Guanine (G) and Cytosine (C).
• They always pair, A with T, and G with C.
• These pairs are called "base pairs".
• A human genome is made of 3,000 million base pairs, split
into 46 chromosomes.
• Almost every cell in our body contains a complete copy of our
genome.

3. Continued…
• A gene is the basic physical and functional unit of heredity.
• Genes, are made up of DNA, and act as to make proteins.
• Proteins carry out most of life’s function.
• When altered causes dysfunction of a protein
• In humans, genes vary in size from a few hundred DNA bases
to more than 2 million bases.

4. Continued…
• The Human Genome Project has estimated that humans have
between 20,000 and 25,000 genes.
• Only about 3% of the DNA actually codes for genes; the rest is
often called "non-coding DNA" because its function is
unknown.
• The first complete human genome was only decoded in 2007.

5. Continued…
• Every person has two copies of each gene, one inherited from
each parent.
• Most genes are the same in all people, but a small number of
genes (less than 1 percent of the total) are slightly different
between people.
• Alleles are forms of the same gene with small differences in
their sequence of DNA bases.
• These small differences contribute to each person’s unique
physical features.

7. Genetic Basis of Human Diseases
Based on their genetic contribution, human diseases can be
classified as
• Monogenic,
• Chromosomal,
• Multifactorial.
Monogenic diseases
• are caused by alterations in a single gene,
• and they segregate in families according to the traditional
Mendelian principles of inheritance.

8. Continued…
Chromosomal diseases
as their name implies, are caused by alterations in chromosomes.
• For instance, within an individual's genome, some
chromosomes may be missing,
• extra chromosome copies may be present, or
• certain portions of chromosomes may be deleted or duplicated.

9. Continued…
Multifactorial
• The vast majority of human diseases can be categorized as
multifactorial.
• These conditions are also referred to as complex diseases,
• They are responsible for most of the burden on our health care
system.

10. Continued…
• Examples of these conditions include
• cardiovascular disease,
• cancer,
• diabetes,
• a number of birth defects
• and psychiatric disorders.
• By definition, complex diseases are caused by variation in
many genes, and they may or may not be influenced by
environment.

11. Need for genetic Research
• The goal of genetic research is to better understand the
mechanisms of disease so that new treatment approaches and
preventative measures can be proposed.
• Now it is possible with the aid of latest technology to identify
the genes responsible for disease and its exact location.
• With this knowledge various therapeutic approaches can be
undertaken to cure the disease.

12. Continued…
• Any particular disease can be approached at the following
levels:
1. At the level of genes encoding the defective protein,
2. At the level of that particular protein causing diseases,
3. And the secondary effects caused by the entry of defective
protein into the cellular machinery.

13. What is Gene Therapy?
• Gene therapy is the application of genetic principles in the
treatment of human disease.
• Gene therapy typically involves the insertion of a functioning
gene into cells to correct a cellular dysfunction or to provide a
new cellular function (Culver, 1994).
• GT is used to correct a deficient phenotype so that sufficient
amounts of a normal gene product are synthesized  to
improve a genetic disorder.

14. Continued…
For example, diseases such as
• Cystic fibrosis,
• Severe combined immunodeficiency syndrome,
• Muscular dystrophy,
• Hemophilia, and
• many cancers result from the presence of defective genes.
• Gene therapy can be used to correct or replace the defective
genes responsible.

15. Continued…
• Gene therapy is not a new idea.
• In 1963, Joshua Lederberg wrote:
• “The ultimate application of molecular biology would be the
direct control of nucleotide sequences in human
chromosomes,. . . . It will only be a matter of time . . . before
polynucleotide sequences can be grafted by chemical
procedures onto a virus DNA.”

16. Continued…
• Less than 30 years later, the first clinical study using gene
transfer was reported (Rosenberg et al., 1990).
• Rosenberg and his colleagues used a retroviral vector to
transfer the neomycin resistance marker gene into tumor-
infiltrating lymphocytes obtained from patients with metastatic
melanoma.

17. Variety of Gene Therapy
Monogenic gene therapy
• Provides genes to encode for the production of a specific
protein
• Cystic fibrosis, Muscular dystrophy, Sickle cell disease,
Haemophilia,
• SCID
Suicide gene therapy
• Provide ‘suicide’ genes to target cancer cells for destruction
• Cancer

18. Continued…
Antisense gene therapy
• Provides a single stranded gene in an ’antisense’
(backward) orientation to block the production of harmful
proteins
• AIDS/HIV

19. Types of Gene Therapy
Somatic gene therapy can be broadly split into two categories:
Ex vivo: which means exterior (where cells are modified outside
the body and then transplanted back in again).
• Cells from the patient’s blood or bone marrow are removed
and grown in the laboratory.
• The cells are exposed to the virus that is carrying the desired
gene.

20. Continued…
• The virus enters the cells and inserts the desired gene into the
cells’ DNA.
• The cells grow in the laboratory and are then returned to the
patient by injection into a vein.
• This type of gene therapy is called ex vivo because the cells
are treated outside the body.

22. In Vivo
In vivo: which means interior (where genes are changed in cells
still in the body).
• During In vivo gene transfer, the genes are transferred directly
into the tissue of the patient.
• In vivo techniques usually utilize vectors.
• Virus is the carrier of desired gene.
• Virus is usually modified to disable its ability to cause
disease.( E.g. deletion of env gene).

23. Continued…
• Viral methods have proved to be the most efficient to date.
• Many viral vectors can integrate the desired gene into the
target cell’s genome.
• Viruses are highly evolved natural vectors for the transfer of
foreign genetic information into cells.
• But to improve safety, they need to be replication defective.

24. Approaches to gene therapy
Several approaches to gene therapy are being tested, including: -
1. Replacing a mutated gene that causes disease with a healthy
copy of the gene.
2. Inactivating, or “knocking out,” a mutated gene that is
functioning improperly.
3. Introducing a new gene into the body to help fight a disease.

25. Continued…
Vector systems can be divided into:
• Viral Vectors: Gene transfer mediated by viral vectors is
referred to as transduction.
• Non-viral Vectors: Gene transfer mediated by non-viral vectors
is referred to as transfection.

26. General approaches used in gene therapy
Non Viral Viral Based
Naked/plasmid DNA
Retrovirus
Adenovirus
Lipofection
Adeno-associated Virus
Gene Gun Lenti Virus
Herpes simplex Virus

27. In vivo versus ex vivo
• In vivo = delivery of genes takes place in the body
• Ex vivo = delivery takes place out of the body, and then cells
are placed back into the body

28. Viral Vectors
Adenovirus
Adenoviruses are:
• medium-sized (90–100 nm),
• Non-enveloped (naked),
• Icosahedral viruses
• Composed of a nucleocapsid and a double-stranded linear
DNA genome.
• There are over 51 different serotypes in humans, which are
responsible for 5–10% of upper respiratory infections in
children, and many infections in adults as well.

29. Continued…
• The genetic material of the adenoviruses is not incorporated
(transient) into the host cell's genetic material.
• The DNA molecule is left free in the nucleus of the host cell,
and the instructions in this extra DNA molecule are
transcribed just like any other gene.

30. Continued…
• The only difference is that these extra genes are not replicated
when the cell is about to undergo cell division so the
descendants of that cell will not have that extra gene.

32. Adeno-associated Viruses
• Adeno-associated viruses, from the parvovirus family, are
small viruses with a genome of single stranded DNA.
• These viruses can insert genetic material at a specific site on
chromosome 19 with near 100% certainty.
• They do not cause any known disease and doesn't trigger
patient immune response.

33. Continued…
• AAV has Low information capacity (it can carry small amount
of DNA).
• Gene is always "on" so the protein is always being expressed,
possibly even in instances when it isn't needed.
• Several trials with AAV are on-going or in preparation, mainly
trying to treat muscle and eye diseases.
• This is possible because AAV viruses can infect non-dividing
(quiescent) cells, such as neurons.

34. Herpes Simplex Viruses
• Herpes simplex viruses (HSV) belong to the subfamily of
Alphaherpesvirinae.
• HSV consists of a relatively large double-stranded DNA 150
kb in length, encased within an icosahedral capsid, which is
wrapped in a lipid bilayer envelope.
• The envelope is joined to the capsid by means of a tegument.
This complete particle is known as the virion.

35. HSV

36. Continued…
The unique features of HSV derived vectors are:
• Very high transgenic capacity of the virus particle allowing to
carry long sequences of foreign DNA,
• the genetic complexity of the virus genome, allowing to generate
many different types of attenuated vectors possessing oncolytic
activity,
• and the ability of HSV vectors to invade and establish lifelong
non-toxic latent infections in neurons from sensory ganglia.

37. Lentiviruses
• Lentivirus is a genus of the Retroviridae family, characterized
by a long incubation period.
• Lentiviruses can deliver a significant amount of genetic
information into the DNA of the host cell, so they are one of
the most efficient methods of a gene delivery vector.
• HIV, SIV, and FIV are all examples of lentiviruses.
• Lentiviral infection have advantages over other gene therapy
methods including high-efficiency infection of dividing and
non-dividing cells, long-term stable expression of a transgene,
and low immunogenicity.

38. Retroviruses
• A retrovirus is any virus belonging to the viral family
Retroviridae.
• All The genetic material in retroviruses is in the form of RNA
molecules.
• When a retrovirus infects a host cell, it will introduce its RNA
together with some enzymes into the cell.
• The retrovirus goes through reverse transcription using reverse
transcriptase and RNA.

39. Continued…
• the double stranded viral genome integrates into the human
genome using integrase
• integrase inserts the gene anywhere because it has no specific
site.
• May cause insertional mutagenesis
• One gene disrupts another gene’s code (disrupted cell division
causes cancer from uncontrolled cell division)
• vectors used are derived from the human immunodeficiency
virus (HIV) and are being evaluated for safety

40. Risk Factors with Viral Vectors
• The concept of gene therapy seems straightforward, but this is
clearly an oversimplification.
• when viral vectors are used to carry genes into the body, they
might infect healthy cells as well as cancer cells
• new gene might be inserted in the wrong location in the DNA,
possibly causing harmful mutations to the DNA or even cancer.
• DNA could unintentionally be introduced into the patient’s
reproductive cells. This could produce changes that may be passed
on if a patient has children after treatment.

41. Continued…
• The possibility that transferred genes could be overexpressed,
leading to too much production of the missing protein as to be
harmful,
• the viral vector could cause an immune reaction.
• The virus could be transmitted from the patient to other
individuals or into the environment.

42. Non-Viral Vectors
Non-viral methods present certain advantages over viral methods,
• simple large scale production and low host immunogenicity.
• Previously, low levels of transfection and expression of the
gene held non-viral methods at a disadvantage;
• however, recent advances in vector technology have yielded
molecules and techniques with transfection efficiencies similar
to those of viruses.

43. Continued…
Naked DNA
• This is the simplest method of non-viral transfection.
• Clinical trials carried out of intramuscular injection of a naked
DNA plasmid have occurred with some success;
• however, the expression has been very low in comparison to
other methods of transfection.

44. Continued…
Electroporation:
• Electroporation designates the use of short high-voltage pulses
to overcome the barrier of the cell membrane.
• can be used to load cells with a variety of different molecules,
like DNA transfer.

45. Lipoplexes and polyplexes
• Lipoplexes and polyplexes, have the ability to protect the DNA
from undesirable degradation during the transfection process.
• Plasmid DNA can be covered with lipids in an organized
structure like a micelle or a liposome.
• When the organized structure is complexed with DNA it is
called a lipoplex.
• Complexes of polymers with DNA are called polyplexes.

46. Continued…
• Cheaper than viruses.
• No immune response.
• The most common use of lipoplexes has been to supply
activated tumor suppressor control genes in the cell and
decrease the activity of oncogenes.
• lipoplexes are useful in transfecting respiratory epithelial cells,
so they may be used for treatment of genetic respiratory
diseases such as cystic fibrosis.
• 100-1000 times more plasmid DNA needed for the same
transfer efficiency as for viral vector.

47. DNA liposome
complexes

49. Continued…
Gene Gun
Also known as a biolistic particle delivery system,
• originally designed for plant transformation,
• is a device for injecting cells with genetic information.
• The payload is an elemental particle of a heavy metal coated
with plasmid DNA.
• This technique is often simply referred to as bioballistics or
biolistics.
• Gene guns have also been used to deliver DNA vaccines.

50. Continued…
• Gene gun make use of the sticky nature of DNA or RNA when
they adhere to biologically inert particles like metal atoms.
• DNA is introduced by accelerating the DNA-Particle complex
in partial vacuum.
• Target tissue is placed in the acceleration path.
• Gene gun is used to deliver a nucleic acid-based hepatitis B
vaccine to both mice and humans, and is presently in clinical
trials (Mumper, 2001).

52. Antisense Oligonucleotides
Antisense Oligonucleotides :
• The use of synthetic oligonucleotides in gene therapy is to
inactivate the genes involved in the disease process.
• Use of antisense oligonucleotides specific to the target gene to
disrupt the transcription of the faulty gene.
• Antisense Oligonucleotides are unmodified or chemically
modified ssDNA, RNA or their analogs.

53. Continued…
Some examples of Antisense Oligonucleotides:
• Peptide nucleic acids (PNAs),
• Locked nucleic acid (LNA),
• Tricyclo-DNA (tcDNA),
• siRNA.

54. Peptide Nucleic Acids(PNAs)
• PNA was first introduced by Nielsen and coworkers in 1991.
• They are electrostatically neutral molecules.
• In PNAs the deoxyribose phosphate backbone is replaced by
polyamide linkages, which is composed of repeating N-(2-
aminoethyl)-glycine units.
• PNAs can form very stable duplexes or triplexes with nucleic
acids.

55. Locked Nucleic Acid (LNA)
• LNA was synthesized by Jesper Wengel in 1998.
• The ribose ring is connected by a methylene bridge (orange)
between the 2’-O and 4’-C atoms thus “locking” the ribose
ring in the ideal conformation for Watson-Crick binding.

56. Continued…
• When incorporated into a DNA or RNA oligonucleotide, LNA
makes the pairing with a complementary nucleotide strand
more rapid and increases the stability of the resulting duplex.
• As a result, LNA oligonucleotides exhibit unprecedented
thermal stability when hybridized to a complementary DNA or
RNA strand.
• LNA based hepatitis C drug called Miravirsen, targeting miR-
122, which is in Phase II clinical testing as of late 2010.

57. Tri-cyclo DNA (tc-DNA)
• Much as LNA, tricyclo-DNA has been designed as a
conformationally constrained oligonucleotide analogue.
Chemically, tc-DNA deviates from natural DNA by three
additional C-atoms between C(5’) and C(3’).
•Increased stability towards nucleolytic degradation.
•Increased RNA affinity.

58. RNA interference (RNAi) :
• RNAi is an antisense mechanism that involves using small
interfering RNA, or siRNA, to target a mRNA sequence. With
siRNA, the cell utilizes a protein complex called RNA-induced
silencing complex (RISC) to destroy the mRNA, thereby
preventing the production of a disease-causing protein.
• Applications of RNAi :
• Cancer
• HIV
• Cardiovascular and Cerebrovascular Diseases
• Neurodegenerative Disorders

60. Human artificial chromosome
• A human artificial chromosome (HAC) is a microchromosome
that can act as a new chromosome in a population of human
cells.
• Instead of 46 a cell can have 47 chromosomes with the 47th
being very small, roughly 6-10 megabases in size, and able to
carry newly introduced genes
• researchers could integrate different genes that perform a
variety of functions, including disease defense.

61. Continued…
• The genetic material introduced by the vectors not only leads
to different expression levels, but the inserts also disrupt the
original genome.
• HACs differ in this regard, as they are entirely separate
chromosomes. This separation from existing genetic material
assumes that no insertional mutants would arise.
• stability and accuracy makes HACs preferable to other
methods such as viral vectors

62. Continued…
• I n 2010, a human artificial chromosome called 21HAC was
reported.
• 21HAC is based on a stripped copy of human chromosome 21
that is mitotically stable.
• Using 21HAC, researchers were able to insert a herpes
simplex virus- thymidine kinase coding gene into tumor cells.
• This "suicide gene" is required to activate many antiviral
medications.
• These targeted tumor cells were successfully, and selectively,
terminated by the antiviral drug ganciclovir.
• This research opens a variety of opportunities for using HACs
in gene therapy.

63. Severe Combined Immunodeficiency
SCID is a primary immune deficiency
• The defining characteristic is usually a severe defect in both
the T- & B-lymphocyte systems.
• Gene is located on chromosome 22 is inherited as an X-linked
recessive disease, which for all practical purposes affects only
boys.
• results in the onset of one or more serious infections within the
first few months of life.
• SCID is caused by an Adenosine Deaminase Deficiency
(ADA)

64. The Case of David Phillip Vetter
• SCID is often called “bubble boy disease”.
• SCID became widely known during the 1970′s and 80′s, when
the world learned of David Vetter,
• A boy with SCID, who lived for 12 years in a plastic, germ-
free bubble.

65. Continued…
• Immediately after being delivered David entered the plastic
germ-free environment that would be his home for most of his
life.
• After being placed in the sterile chamber, David was touched
only through special plastic gloves attached to the walls of the
chamber.
• David later received a bone marrow transplant from his sister,
Katherine. Following that, David became ill.

66. Continued…
• He died 15 days later, from Burkitt's lymphoma at the age of
12.
• The autopsy revealed that the donor’s bone marrow contained
traces of dormant Epstein-Barr virus which was undetectable
in the pre-transplant screening.
• Once transplanted, the virus spreaded and produced hundreds
of cancerous tumors.

67. Continued…
• Dr. W. French Anderson presides over the first experimental
use of a gene therapy in 1990. The patient was a four-year old
girl name Ashanti DeSilva.
• She was born with SCID.
• Dr. Anderson’s team drew blood from their patient, and
replaced the defective gene with a functional variant.
• The therapy partially restores Ashanti’s immune system.

68. Continued…
• It temporarily stimulates production of the missing enzyme,
but does not generate new cells with functional genes.
• Ashanti continues to receive injections of corrected T-cells
every two months.
• She also takes doses of the enzyme itself, in the form of a drug
called PEG-ADA.
• She is able to lead a normal life.

69. Ornithine transcarbamylase deficiency
• Gene therapy had been considered a possibility for curative
treatment for OTC deficiency.
• OTC deficiency is the most common urea cycle disorder in
humans.
• OTC deficiency is inherited in an X-linked recessive manner.
• In severely affected individuals, ammonia concentrations
increase rapidly causing ataxia, lethargy and death without
rapid intervention.

70. Continued…
• First case of death due to gene therapy was of Jesse Gelsinger.
• He was suffering from OTC.
• Jesse received gene therapy via adenovirus vector.
• Soon after the therapy Jesse developed severe inflammatory
reactions due to massive release of interleukin in response to
adenovirus.
• He died soon thereafter.
• Currently, the only option for curing OTC deficiency is a liver
transplant, which restores normal enzyme activity.

71. Few more examples…
• Single gene disorder are the most suitable candidate for gene
therapy.
• Ex;
• Cystic fibrosis: adenovirus administered via aerosol delivery
into nasal passages and lungs.
• Only transient expression observed  because adenovirus
does not integrate into genome like retroviruses.
• Familial Hypercholesterolemia: Retroviral vector used to
infect liver cells (~15% of patients liver) ex vivo.
• Infused back into patient.
• Improvement has been seen.
• is used in many trials since then.

72. Continued…
HIV:
• University of Pennsylvania researchers have successfully
genetically engineered the immune cells of 12 HIV positive
patients to resist infection, and decreased the viral loads of
some patients taken off the antiretroviral drug therapy (ADT)
entirely.
• By inducing the mutations, the scientists reduced the
expression of CCR5 surface proteins. Without those, HIV
cannot enter, rendering the patients’ cells resistant to infection.
• The study also shows promise in the approach’s ability to
suppress the virus.

73. How stem cells and gene therapy might
work together
1. A sample of bone marrow is
removed.
2. Stem cells are isolated and
allowed to multiply in culture.
3. Cells are treated with a modified
virus containing a therapeutic
gene

74. 1. The virus is taken up by individual
cells and the therapeutic gene goes
into the cell's nucleus.
2. Treated ("corrected") cells are
injected into the bloodstream.
3. Treated cells respond to injury signals
from degenerating muscle or other
tissues and migrate out of the
bloodstream.
4. Treated cells patch damage and build
healthy tissue.

75. Limitations of Gene Therapy
Short Lived:
• It is Hard to rapidly integrate therapeutic DNA into genome
and rapidly dividing nature of cells prevent gene.
• Have to give multiple rounds of therapy.
Gene delivery:
• Dependence on cell cycle by some viral vectors (i.e.
mitosis required)
• Most viral vectors are unable to accommodate full length
human genes containing all of their original regulatory
sequences
• Random integration can adversely affect expression

76. Continued…
Duration of gene activity:
• Non-integrating delivery will be transient (transient
expression)
• Integrated delivery will be stable
Patient safety:
• Immune hyper-responsiveness (hypersensitivity reactions
directed against viral vector components or against
transgenes expressed in treated cells).
• Integration is not controlled  oncogenes may be
involved at insertion point, can lead to cancer.
• Viruses may also cause disease once inside

77. Continued…
Multigene Disorders:
• Heart disease, high blood pressure, Alzheimer’s, arthritis and
diabetes are hard to treat because one need to introduce more
than one gene
• May induce a tumor if integrated in a tumor suppressor gene
because of insertional mutagenesis.
One problem with gene therapy is that one does not have control
over where the gene will be inserted into the genome.

78. Continued…
• The location of a gene in the genome is of importance for the
degree of expression of the gene and for the regulation of the
gene (the so-called "position effect"), and
• thus the gene regulatory aspects are always uncertain after
gene therapy .
Expense:
• Costly because of cell culturing needs involved in ex vivo
techniques.
• Virus cultures for in vivo delivery.

79. Recent Developments
• Genes introduced into CNS using liposomes coated in
polymer have the potential for treating Parkinson’s disease.
• RNA interference or gene silencing to treat Huntington’s
disease
• Introduction of normal frataxin gene in animal models of
friedreich’s ataxia with the help of AAV has shown promise.
• Gene therapy leads to robust improvements in animal model of
a fatal congenital pediatric disease known as X-linked
myotubular myopathy.

80. Continued…
• According to a recent study delivering a single injection of
gene therapy to the spinal cord of rats following injury
promotes the survival of nerve cells and improves hind limb
function within weeks.
• The effects of gene Cyclin A2 (CCna2) using gene therapy as a
tool for cardiac regeneration, could lead to a viable treatment
option for patients who suffer from myocardial infarction, or
heart attack.
• New gene therapy proves promising as hemophilia treatment.

81. Conclusion
• As the science and technology will advance new therapeutic
ways will always be discovered.
• Gene therapy has a very wide application.
• Almost every human disease has a genetic basis,
-but with the current limitations of delivery vectors and the
limited knowledge of human DNA,
-it will take a long time to develop designer therapies for the
patients.
• By using gene therapy one day most of the diseases will be
diagnosed before birth and cured.
• Ultimately someday gene therapy will take human race to the
pinnacle of evolutionary excellence.