gene therapy for masters and bachelors

1. Gene Therapy
Gene therapy is the use of DNA
pharmaceutical agent to treat disease
as
a

2. Gene therapy
Gene therapy can be broadly defined as the transfer of
genetic material into a cell to transiently or permanently
alter the cellular phenotype.
Molecular surgery

3. What is Gene Therapy
• It is a technique for correcting defective genes
that are responsible for disease development
• There are four approaches:
1. A normal gene inserted to compensate for a
nonfunctional gene.
2. An abnormal gene expression suppressed (antisense Tech)
3. An abnormal gene repaired through selective
reverse mutation
4. Change the regulation of gene pairs

4. Gene Therapy Vs Conventional Therapy
Gene Therapy
Conventional Therapy
Materials
DNA, RNA; Cells, Tissues, Or Organs.
Small molecules, Peptide,
Proteins.
Delivery
Usually required to be delivered into cells
(antisense ODN) or Nucleus (genes).
Effect on the cell membrane
or diffuse into cells
Mechanisms
Usually cure the causes of the diseases
Usually relieve the
symptoms or signs
Duration of
Effect
Can be permanent and also can be passed
down to next generation in germline gene
therapy.
Usually stop the effect once
stop taking it.
Ethics
Major Issues
Usually Not

5. Purpose & approach 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
•
Antisense gene therapy
• Provides a single stranded gene in an’antisense’ (backward)
orientation to block the production of harmful proteins
• AIDS/HIV

6. Barriers Of Gene Delivery

7. Strategies for Transgene Delivery
Ex Vivo
Cells removed from body
Transgene delivered
Cells cultured
Cells returned to the body
In Vivo
Transgene delivered
directly into host

8. Gene Therapy Principles
AAV
Nucleus
Adenovirus
Therapeutic
Protein
Retrovirus/Lentivirus
Naked DNA
Target
Cell

9. Types of vectors for gene delivery
• RNA viruses (Retroviruses)
1. Murine leukemia virus (MuLV)
2. Human immunodeficiency viruses (HIV)
3. Human T-cell lymphotropic viruses
(HTLV)
• DNA viruses
1. Adenoviruses
2. Adeno-associated viruses (AAV)
3. Herpes simplex virus (HSV)
4. Pox viruses
5. Foamy viruses
• Non-viral vectors
1. Liposomes
2. Naked DNA
3. Liposome-polycation complexes
4. Peptide delivery systems

10. Viral Vectors: Gene + Protein
Coat
• Disabled viral vectors
– Genes that cause disease are removed
– Gene of interest is inserted
• Altered virus should transfer helpful genes to cells but should not
multiply or produce disease
• Viruses bind to the cell surface receptors of cell membrane
and deliver its genetic contents
– Do DNA viruses, RNA viruses or both enter the nucleus?
• The cell will use the inserted gene to produce a therapeutic
protein

11. Retrovirus for gene delivery

12. 1. Modified Retroviruses (RNA viruses)
(1 of 2)
Advantages
• Good at inserting genes into host
chromosome
-
Used with partial success treating Gaucher’s disease
Successfully cured 4 babies of S.C.I.D.S. in early 2000
• Severe Combined Immunodeficiency Syndrome
(Bubble Baby)

13. 1. Modified Retroviruses (RNA viruses)
(2 of 2)
Disadvantages
1. Inserts genes randomly. Possible
Consequences?
2. Usually needs an actively dividing host cell
• Therefore, not used for Cystic Fibrosis
1. Modified virus may mutate and cause
serious disease.

14. 3-D visualization of retrovirus structure.

15. Life cycle of a retrovirus
Gene therapy
constructs
maintained at
this stage.

16. Retrovirus genome
Encapsidation
(packaging)
16

18. Retrovirus genome
Encapsidation
(packaging)
Retrovirus vector construction for gene therapy
5’ LTR Packaging Gene X
Neor
3’ LTR
18

19. Engineering a virus into a viral vector
http://www.edu365.com/aulanet/comsoc/Lab_bio/simulacions/GeneTherapy/GeneTherapy.htm
Therapeutic
Packaging
gene
Vector DNA
wildtype virus
Viral vector

20. Gene transfer
Y
vector
Vector uncoating
Episomal vector
Target cell
Integrated expression
cassette
Therapeutic mRNA
and protein

21. Adenovirus

22. Adenovirus particle structure:
• Nonenveloped
particle
• Contains linear
double stranded DNA
• Does not integrate
into the host genome
• Replicates as an
episomal element in
the nucleus

24. Herpes Simplex Virus
Advantages
• Large insert size
• Could provide long- term CNS gene expression
• High titer
Disadvantages
• System currently under development
• Current vectors provide transient expression
• Low transduction efficiency

25. Non-viral vectors
1. Liposomes
2. Naked DNA

26. Naked DNA
• Biolistics now used routinely. DNA coated
particles are literally blasted into cells by an
explosive discharge.
• Electroporation
• Pronuclear microinjection
26

27. ‘Particle Gun’
27

28. ‘Particle Gun’
• DNA coated on pellets
is forced down the
barrel of a ‘Particle
Gun’ by an explosive
charge
• The particles are forced
through the cell wall
where the DNA is
released
Petri Dish
with cultures
Explosive
Charge
Projectile
DNA coated
pellets
Barrel
Vent
Stop plate

29. Nano particles for gene
delivery
The electrostatically coated poly(beta-amino ester)
nanoparticles can facilitate ligand-mediated gene
delivery.

30. The more promising polymers for gene delivery is
degradable poly(beta-amino ester), 1,3-diaminopentaneterminated
poly(5-amino-1-pentanol-co-1,4-butanediol
diacrylate) (C32-117).
This polymer functions by binding to and protecting DNA
from degradation, enabling efficient cellular uptake, and
enabling subsequent intracellular endosomal escape.
However, as with many nanoparticle formulations, its
systemic use in vivo is limited due to poor biodistribution and
lack of tissue-specific targeting

31. Cationic nanoparticles are formed by first complexing poly (b-amino ester) C32-117
with plasmid DNA at a 30:1 polymer:DNA weight/weight ratio (w/w).
These nanoparticles are then coated with poly(glutamic acid)-based peptides (poly-E
or poly-E-cat) at 2.5:1–20:1 peptide:DNA w/w.
Variation in peptide w/w tunes the biophysical properties of the nanoparticles and
subsequent localization of gene delivery by the nanoparticles in vivo.

32. Pronuclear microinjection of DNA

33. Electroporation

34. What is electroporation?
• A short controlled pulse of electricity to cell
momentarily disrupting lipid bilayer.
• Small pores (40-120nm) reseal quickly.
Cell wall
Nucleu
s
DNA enters

35. Electroporation

36. Electroporation
• Use of high-voltage electric shocks to introduce DNA into cells
• Cell membranes: electrical capacitors unable to pass current
• Voltage results in temporary breakdown and formation of
pores
Harvest cells and resuspend in electroporation buffer
Add DNA to cell suspension…for stable transfection
DNA should be linearized, for transient the DNA may
be supercoiled
electroporat
e
Selection process for
transfectant

37. This electroporator is for low-current applications
such as those using small electrodes

38. Ex vivo Electroporation

39. Liposomes

40. Lipofection (or liposome transfection) is a technique used to inject
genetic material into a cell by means of liposomes, which are vesicles
that can easily merge with the cell membrane since they are both made
of a phospholipid bilayer.
Lipofection generally uses a positively charged (cationic) lipid to form
an aggregate with the negatively charged (anionic) genetic material.
A net positive charge on this aggregrate has been assumed to increase
the effectiveness of transfection through the negatively charged
phospholipid bilayer.
This transfection technology performs the same tasks as other
biochemical procedures utilizing polymers, DEAE dextran, calcium
phosphate, and electroporation. The main advantages of lipofection are
its high efficiency, its ability to transfect all types of nucleic acids in a
wide range of cell types, its ease of use, reproducibility, and low
toxicity.

41. Lipofection (or liposome transfection)

46. Example:

47. Gene therapy for silencing
un wanted gene expression
Antisense technology

48. Antisense technology
A single-stranded RNA or DNA molecule that is complementary to a
target mRNA pairs with the mRNA and prevents translation.
This strategy works well in the laboratory on cultured cells and on model
organisms.
Clinical example: treatment against cancers. The tumor sizes decreased
but this was mainly due to the production of interferons in response to
high doses of foreign RNA. If the dose was lowered to prevent the
interferon response, the clinical benefits largely disappeared as well.

49. Antisense technology

50. SiRNA

51. SiRNA is small interfering RNA. It is also abbreviated as RNAi – RNA
intereference.
1.Long double stranded RNA’s will be cleaved by an enzyme called Dicer
(endoribonuclease) into short double stranded fragments (-20-25
nucleotides) called siRNA
2. Double stranded siRNA will then be separated into single stranded RNA’s.
one strand is called the “guide strand” and the other “Passenger strand”. The
guide strand will further bind to RISC (RNA-induced silencing complex) and
the passenger strand is degraded. Sometimes, RISC can also be called as
RITS (RNA-induced transcriptional silencing). Originally, thought to be an
ATP-dependent
helicase, which is responsible for unwinding and
degradation of the passenger strand, however, later on found to be ATPindependent and the protein components of the RISC does this.
3. Activated RISC complex locates complementary mRNA’s within the cell.
4. Now, this siRNA+RISC complex will go and bind to the complementary
bases in the mRNA strand of the targeted gene. “Argonaute”, the catalytic
components (protein components) of the RISC will then causes the targeted
mRNA strand to cleave, therefore blocking the protein synthesis.

52. How does siRNA
work ??????
(fig.cox.miami.edu/.../gene/how_siRNA_works.htm)

54. Inherited Disease

55. Inherited Disease
A large number of diseases are known to be inherited from the parents to
the offspring. Such diseases are known as Inherited Diseases.
A large number of diseases are known to be inherited from the parents to
the offspring. Such diseases are known as genetic diseases. Most of these
diseases are caused by the expression of recessive genes.
The genetic diseases can be broadly classified into two types:
•Autosomal disorders
•Allosomal disorders
Autosomal Disorders: These are metabolic disorders caused by the
expression of some genes present on somatic chromosomes. Such
disorders express equally in both the sexes.
Allosomal Disorders: hese disorders are caused by genes present on the
sex chromosomes. The abnormal disorders express more commonly in
males than females

56. Gene therapy for inherited diseases are
Severe Combined Immunodeficiency Disease
Ornithine transcarbamylase (OTC) deficiency
Familial Hypercholesterolemia
Cystic Fibrosis
Thalassemia
Lesch-Nyhan syndrome
Hunter’s syndrome
Sickle cell trait and Sickle cell anemia

57. Severe Combined Immunodeficiency Disease (SCID)
• SCID is caused by an Adenosine Deaminase Deficiency (ADA)
– Gene is located on chromosome #22
(32 Kbp, 12 exons)
– Deficiency results in failure to develop functional T and B
lymphocytes
– ADA is involved in Adenine degradation
– Lack of ADA leads to a 100-fold increase in the cellular
concentration of dATP, a strong inhibitor of ribonucleotide
reductase .
– High levelsof dATP produce a general deficiency of other dNTPs
in T lymphocytes.
– Accumulation of nucleotide metabolites = TOXIC to developing T
lymphocytes
– B cells don’t mature because they require T cell help
– Patients cannot withstand infection  die if untreated

58. The first case for gene therapy in the world is SCID
AMP
IMP
ADA gene mutation
adenosine ADA inosin
e
adenosine
dAMP
(adenosine deaminase )
ADA
dADP
dATPInhibit nucleotide reductase
T, B cell proliferation (-)
Severe Combined ImmunoDeficiency

59. Severe Combined Immunodeficiency
Disease (SCID)
• September 14, 1990 @ NIH, French Anderson and R.
Michael Blaese perform the first GT Trial
– Ashanti (4 year old girl)
• Her lymphocytes were gene-altered (~109) ex vivo 
used as a vehicle for gene introduction using a
retrovirus vector to carry ADA gene (billions of
retroviruses used)
– Cynthia (9 year old girl) treated in same year
• Problem: WBC are short-lived, therefore treatment must
be repeated regularly

60. Retrovirus used to deliver gene for Adenosine deaminase
Gene therapy
constructs
maintained at
this stage.

61. Ornithine transcarbamylase (OTC)
deficiency
– Ornithine transcarbamylase (OTC) deficiency
• Urea cycle disorder (1/10,000 births)
• Encoded on X chromosome
– Females usually carriers, sons have disease
– Urea cycle = series of 5 liver enzymes that rid the body
of ammonia (toxic breakdown product of protein)
• If enzymes are missing or deficient, ammonia
accumulates in the blood and travels to the brain
(coma, brain damage or death)

63. Ornithine transcarbamylase (OTC)
deficiency
• Severe OTC deficiency
– Newborns  coma within 72 hours
• Most suffer severe brain damage
• ½ die in first month
• ½ of survivors die by age 5
– Early treatment
• Low-protein formula called “keto-acid”
– Modern day treatment
• Sodium benzoate and another sodium derivative
• Bind ammonia  helps eliminate it from the body

64. Disorders Associated with Defects
in Receptor Proteins
Familial Hypercholesterolemia
• This commonly results from an autosomal dominant defect
in a gene for the LDL receptor or receptor function.
• At least 900 mutations have been identified affecting
different aspects of LDL uptake, metabolism and regulation.
• De-novo cholesterol synthesis is normally suppressed by
exogenous cholesterol intake; with receptor processing
defects this function is lost and markedly elevated cholesterol
levels result.
• Cholesterol levels are elevated to such an extent that
atherosclerotic disease resulting in fatal cardiovascular events
beginning in the second & third decades .

65. There are five major classes of FH due to LDLR
mutations:
– Class I: LDL receptor (LDL-R) is not synthesized at all
– Class II: LDL-R is not properly transported from the endoplasmic
reticulum to the Golgi apparatus for expression on the cell surface
– Class III: LDL-R does not properly bind LDL on the cell surface (
this may be caused by a defect in either Apolipoprotein B100
or a defect in LDL-R
– Class IV: LDL-R bound to LDL does not properly cluster in clathrincoated pits for receptor-mediated endocytosis
– Class V: the LDL-R is not recycled back to the cell surface

68. • Major issue is LDL receptor mutation
• This data base shows all the different
mutations
• For Familial hypercholesterolemia there are
806 mutations
• 457 mutations are missense and nonsense

69. Substitution mutations
•
•
•
•
GGG-AGG Gly-Arg Hypercholesterolaemia
GCG-GAG Ala-Glu Hypercholesterolaemia
CTC-CCC Leu-Pro Hypercholesterolaemia
cGAG-TAG Glu-Term
Hypercholesterolaemia

70. – Gene Therapy for Familial Hypercholesterolemia
– 1993  First attempt
• Retroviral vector used to infect 3.2 x 109 liver cells (~15%
of patients liver) ex vivo
– Infused back into patient
– Improvement seen
– Has been used in many trials since then

71. Cystic Fibrosis

72. Gene therapy for Cystic Fibrosis
• Cystic fibrosis (CF) is inherited as an autosomal
recessive disease
• CF affects the epithelial cells lining air passages to
the lungs
• CF causes a buildup of mucus in the airways

73. Clinical Features
• Classic cystic fibrosis is characterized by chronic
bacterial infection of the airways and sinuses, fat
maldigestion due to pancreatic exocrine
insufficiency, infertility in males due to obstructive
azoospermia, and elevated concentrations of
chloride in sweat.
• Patients with nonclassic cystic fibrosis have at least
one copy of a mutant gene that confers partial
function of the CFTR protein, and such patients
usually have no overt signs of maldigestion
because some pancreatic exocrine function is
preserved.

74. Gene therapy for Cystic Fibrosis
• In CF, there is a defective ion channel protein = cystic
fibrosis transmembrane conductance regulator
(CFTR)
• CFTR regulates the balance of Chloride ions in
epithelial cell membranes
• Patients with Cystic Fibrosis make an altered version
of this protein
– Protein is misfolded
– What types of proteins are involved in helping
other proteins fold properly?

75. Gene therapy for Cystic Fibrosis
• Adenovirus vector was used to deliver a
normal ion channel protein to airway cells in
a patient’s nose or lungs
• What is special about adenovirus?

76. Thalassemia

77. Gene therapy for
thalassemia
Thalassemia (also spelled thalassaemia) is an inherited
autosomal recessive blood disease.
In thalassemia, the genetic defect which could be either
mutations or deletion results in reduced rate of
synthesis or no synthesis of one of the globin α or βchains that make up hemoglobin.
Reduced synthesis or no synthesis of one of the globin
chains can cause the formation of abnormal hemoglobin
molecules, thus causing anemia, the characteristic
presenting symptom of the thalassemias.

78. The thalassemias are classified according to which chain of the hemoglobin
molecule is affected. In α thalassemias, production of the α globin chain is
affected, while in β thalassemia production of the β globin chain is affected.
β globin chains are encoded by a single gene on chromosome 11; α globin
chains are encoded by two closely linked genes on chromosome 16.
Thus in a normal person with two copies of each chromosome, there
are two loci encoding the β chain, and four loci encoding the α chain. Deletion
of one of the α loci has a high prevalence in people of African or Asian descent,
making them more likely to develop α thalassemias. β thalassemias are
common in Africans, but also in Greeks and Italians.
Beta-thalassemia (β-thalassemia) is a form of thalassemia due to mutations in
the HBB gene on chromosome 11, inherited in an autosomal recessive fashion.
The severity of the disease depends on the nature of the mutation.
•Mutations are characterized as (βo) if they prevent any formation of β chains.
•Mutations are characterized as (β+) if they allow some β chain formation to
occur.
Diagnosis: Screening, Pre-natal diagnostics, check for microcytosis (mean cell
haemoglobin < 27 pg or mean red cell volume < 80 fl).

79. Diagnosis of β-thalassemia Deletion
by Southern Blotting
• Autosomal recessive, decreased or absent β-globin protein.
• Mutant alleles have large deletions or point mutations.
Restriction Enzyme
Cut Sites

80. Gene therapy for Betathalassemia
 Gene transfer of a regulated β-globin gene in
HSCs would reduce the imbalance between aand β-globin chains in erythroid cells
 Transplantation of autologous, genetically
corrected HSCs would represent an alternative
therapy for thalassemic patients lacking a suitable
bone marrow donor

81. TERAPIA GENICAfor β-thalassemia
Gene therapy DELLA ß-TALASSEMIA
21_11.jpg
β-globin vector
Purification of CD34+ cells
Patient
Transduction
Infusion of genetically-corrected cells

82. Lesch-Nyhan syndrome:
X-Linked Recessive Disorders
(HGRPT deficiency)

83.  Lesch-Nyhan syndrome condition is inherited in an Xlinked recessive pattern. It mostly affects male, that they
have only one X chromosome, thus one altered copy of the
gene is sufficient to cause the condition. In females, who
have two X chromosomes, a mutation must usually be
present in both copies of the gene to cause the disorder.
 Lesch-Nyhan syndrome (LNS), also known as Nyhan’s
syndrome, is a rare, inherited disorder caused by a
deficiency
of
the
enzyme
hypoxanthine-guanine
phosphoribosyl transferase (HGPRT) or Kelley-Seegmiller
Syndrome that affects the level of uric acid in the body.
This disease often affects males. Males with this syndrome
develop physical handicaps, mental retardation, and kidney
problems. The symptoms of LNS usually appear between
the ages of 3 and 6 months.

84.  The 3 main features of the disease are:
Excessive production of uric acid
Neurological problems, especially mental retardation
and spastic cerebral palsy
Behavioral disorders- confusion, anxiety, fear, and
obsession

85. Diagnosis
 The diagnosis of Lesch-Nyhan syndrome is based
initially on the distinctive pattern of the child's
symptoms, most commonly involuntary muscle
movements or failure to crawl and walk at the usual
ages.
 In some cases the first symptom is related to
overproduction of uric acid; the parents notice
"orange sand" in the child's diapers. The "sand" is
actually crystals of uric acid tinged with blood.
Measuring the amount of uric acid in a person's blood
or urine can not definitively diagnose Lesch-Nyhan
syndrome. It is diagnosed by measuring the activity
of the HPRT enzyme through a blood test. When the
activity of the enzyme is very low it is diagnostic of
Lesch-Nyhan syndrome.

86. Hunter’s syndrome: Xlinked recessive disorder

87. Hunter’s syndrome, an X-linked recessive disorder.
Hunter syndrome, or mucopolysaccharidosis Type II, is a
lysosomal storage disease caused by a deficient (or absent)
enzyme, iduronate-2-sulfatase.
The syndrome is named after physician Charles A. Hunter
(1873–1955), who first described it in 1917

88. Hunter syndrome, or mucopolysaccharidosis II (MPS II), is a
serious genetic disorder that primarily affects males (X-linked
recessive).
It interferes with the body's ability to break down and recycle
specific
mucopolysaccharides,
also
known
as
glycosaminoglycans or GAG. Hunter syndrome is one of
several related lysosomal storage diseases.
In Hunter syndrome, GAG builds up in cells throughout the
body due to a deficiency or absence of the enzyme
iduronate-2-sulfatase (I2S).
This buildup interferes with the way certain cells and organs
in the body function and leads to a number of serious
symptoms. As the buildup of GAG continues throughout the
cells of the body, signs of Hunter syndrome become more
visible.

89. X-linked recessive
Hunter’s syndrome

90. sickle cell anaemia

91. The genetics of sickle cell anaemia
The shape of the haemoglobin molecule is controlled by two alleles
• Normal Haemoglobin allele
• Sickle Cell Haemoglobin allele
There are three phenotypes
Normal
Normal individuals have two normal haemoglobin alleles
Sickle cell anaemia, a severe form where all the red blood cells are affected.
Sickle cell anaemia patients have two sickle cell alleles in their genotypehomozygous
Sickle cell trait, a mild condition where 50% of the red blood cells are affected.
Sickle cell trait individuals are heterozygotes, having one of each allele

92. Codominant genotypes
Genotypes
HbNHbN
HbNHbS
HbSHbS
Phenotypes
Normal haemoglobin
Sickle cell trait
Sickle cell anaemia

93. The success of gene therapy is based on:
 efficient gene transfer into target cells
 adequate level of transgene expression
 persistence of gene expression
 regulation of gene expression
 tolerance to transgene product
 safety

94. Problems with Gene Therapy
•
Short Lived
– Hard to rapidly integrate therapeutic DNA into genome and rapidly
dividing nature of cells prevent gene therapy from long time
– Would have to have multiple rounds of therapy
•
Immune Response
– new things introduced leads to immune response
– increased response when a repeat offender enters
•
Viral Vectors
– patient could have toxic, immune, inflammatory response
– also may cause disease once inside
•
Multigene Disorders
– Heart disease, high blood pressure, Alzheimer’s, arthritis and
diabetes are hard to treat because you need to introduce more than
one gene
•
May induce a tumor if integrated in a tumor suppressor gene because
insertional mutagenesis

95. Problems Doing Gene therapy (1 of 2)
Inefficient gene delivery—not suitable for all genetic
diseases
1. Most effective if Stem cells are involved
•
•
Only to correct a few cells with the gene
E.g. Blood stem cells: SCIDS and Gaucher Disease
1. Less effective or Ineffective if many cells must be
corrected
•
•
Brain cells (Tay-Sacs disease, Huntington’s disease)
Cystic Fibrosis

96. Problems Doing Gene therapy (2 of 2)
4. Insertion of Gene isn’t always permanent
• e.g. Gaucher Disease: temporary cure until
GCase gene “popped” out of chromosome
4. Insertion of gene into genome could disrupt
other genes.
• Possible consequences?
4. Some viruses elicit immune response or may
cause disease
• E.g. Jesse Gelsinger died in 1999