Gene Therapy, Somatic cell gene therapy, germ line gene therapy, classical gene therapy, non-classical gene therapy, targets of gene therapy, barriers of gene therapy, ex vivo gene therapy, in vivo gene therapy, vectors for gene delivery, antisense therapy

1. PRADEEP SINGH
M.Sc. Medical Biochemistry IIIrd Yr
HIMSR, Jamia Hamdard
New Delhi
04/11/2019
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2.  What is Gene Therapy?
 Types of Gene Therapy
 Barriers of gene delivery
 Strategies for transgene
delivery
 Types of vectors for gene
therapy
 Some diseases treated by
gene therapy
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3.  Gene Therapy is the use of
genetic material (DNA) as a
pharmaceutical agent to
prevent or cure a disease.
 Gene therapy typically aims
to supplement a
defective/mutant allele with
a functional one.
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5.  It is a technique for correcting defective genes that are responsible for a disease.
 It is essentially an intervention that alters the instruction set of a cell.
 There are four approaches:-
1. A normal gene is inserted to compensate for a non-functional/mutated gene.
2. An abnormal gene expression is suppressed.
3. An abnormal gene is repaired through selective reverse mutation.
4. Change the regulation of gene pairs.
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6.  1930’s “Genetic Engineering” - plant/animal breeding
 1960’s First idea of using genes therapeutically
 1950’s-1970’s Gene transfer methods developed
 1970’s-1980’s Recombinant DNA technology
 1990 First gene therapy in human (ADA deficiency)
 Since then, more than 2,500 clinical studies have been initiated for
a broad range of applications, from a variety of monogenic diseases
to infectious diseases, complex neurodegenerative disorders, and
cancer.
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7. Gene Therapy Convential Therapy
Materials DNA, RNA; Cells, Tissues or
organs
Small molecules, Peptide,
Proteins
Delivery Usually required to be
delivered into cells (Antisense
Oligonucleotides) or Nucleus
(genes)
Effect on the cell membrane
or diffuse into cells
Mechanisms Usually cure the cause of the
diseases
Usually relieve the symptoms
or signs of disease
Duration of Effect Can be permanent and also
can be passed down to next
generation in germline gene
therapy.
Usually stop the effect after
stop taking it.
Ethics Major Issues Usually Not 7

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9. A vector delivers the therapeutic gene into a patient’s
target cell
The target cell become infected with the viral vector
The vector’s genetic material is inserted into the target
cell
Functional proteins are produced from the therapeutic
gene causing the cell to return to a normal state
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10. On the basis of class of disease: Genetic disease Vs
complex acquired disorder
By the characteristics of the gene delivery vehicle:
Integrating Vs non-integrating vectors
Whether the vector is administered in vivo (directly into
the patient) or ex vivo (in cultured cells taken from the
patient that are subsequently transplanted back)
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11.  Monogenic gene therapy
 Provides genes for the production of a specific protein
 Cystic fibrosis, Muscular dystrophy, Sickle cell disease, hemophilia, SCID
 Suicide Gene Therapy
 Provide ‘suicide’ genes to target cancer cells for destruction
 Cancer
 Antisense Gene
 Provides a single stranded oligonucleotides in an ‘antisense’ (backward) orientation to
block the production of harmful proteins
 AIDS/HIV
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12.  Somatic cell gene therapy
 Germ line gene therapy
Current gene therapy is exclusively somatic cell gene therapy which involves the
introduction of genes into somatic cells of an affected individual.
The prospect of human germline gene therapy is currently not sanctioned.
Germline gene therapy targets the reproductive cells, meaning any changes made
to the DNA will be passed on to the next generation. The effects of gene therapy are
too unpredictable. Any additional mutations that are introduced as a result will be
passed on to the next generation.
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13. Classical Gene Therapy: Genes are delivered to the
appropriate target cells with the aim of obtaining the
optimal expression of the introduced genes.
Non-classical Gene Therapy: Genes are delivered to
inhibit the expression of genes associated with the
pathogenesis.
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14. 1. Ex vivo Approach
 Rapidly dividing cells such as hematopoietic stem cells are used
 Integrating vectors are used (Retroviral/lentiviral vectors)
2. In vivo approach
 Long lived post-mitotic cells are used (lymphocytes)
 Non-integrating vectors are used (Adenoviruses/AAV)
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16.  Central Nervous System (Huntington’s
Disease)
 Respiratory Tract
 Other solid organs (Liver)
 T cells and Hematopoeitic cells
 Muscles
 Vascular system (Lymphocytes)
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18.  Extracellular
1. Epithelial Barriers;
2. Circulation and The components such as
RBC, serum proteins, Enzymes, etc.
3. Reticuloendothelial cleaning system, etc.
Require inert surface and targeting
molecules for specific transportation of
complex in the blood.
 Intracellular
1. Cell membrane
2. Endosomal Membrane
3. Nuclear Membrane
4. DNA releasing at Right Site and Right Time
Require cationic groups for successful gene
delivery.
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20. Strategies for Transgene Delivery
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Ex Vivo In Vivo
Cells are removed from the body
Transgene delivered into cultures
cells
Cells returned to the body
Transgene delivered directly
into host

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23. RNA viruses (Retroviruses)
1. Murine leukemia virus (MuLV)
2. Human immunodeficiency virus (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. Liposomes-polycation complexes
4. Peptides delivery systems. 23

24.  Disabled viral vectors
– Pathogenicity is attenuated but infection ability is still
maintained.
– 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.
• The cell will use the inserted gene to produce a therapeutic protein
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26. 1. Modified Retroviruses (RNA viruses)
Advantages
 Good at inserting genes into host chromosome
–Used with partial success treating Gaucher’s disease
–Successfully cured 4 babies of SCID in early 2000
[Severe Combined Immunodeficiency Syndrome (Bubble baby)]
Disadvantages
1. Insert genes randomly.
2. Usually needs an actively dividing host cell to enter
Therefore, not used for Cystic Fibrosis
3. Modified virus may cause mutation and serious disease.
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Gag: processed to matrix and other core proteins that determine retroviral core.
Pol: reverse transcriptase, RNase H and integrase functions.
Env: envelope protein, resides in lipid layer; determine viral protein

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32. 325’ LTR Packaging Gene X Neor 3’ LTR
Insertion of new gene

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Vector DNA
Helper DNA
wildtype virus
Viral vector
replication
proteins
structural
proteins
Packaging
Therapeutic
gene
essential viral genes
Packaging cell

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vector
Vector uncoating
Therapeutic mRNA
and protein
Episomal vector
Integrated expression
cassette
Target cell

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36.  Non-enveloped virus
 Contains linear double stranded
DNA
 Can enter both dividing as well as
non-dividing cells.
 Does not integrate into the host
genome
 Replicates as an episomal
element in the nucleus
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39. AAV is a simple, non-pathogenic, single stranded DNA
virus dependent on the helper virus (usually adenovirus)
for replication.
AAV enters the host cell, becomes double stranded and
gets integrated into host genome.
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41. 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 41

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43. Non-viral vectors
1. Naked DNA
2. Liposomes
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45.  It is simplest method of injecting naked DNA into the human
body.
 Relatively low level of expression and but sufficient for use in
DNA vaccination.
 Techniques commonly used for naked DNA delivery:
1. Biolistics (or gene gun) now used routinely. DNA coated
particles are literally blasted into cells by an explosive
discharge.
2. Electroporation
3. Pronuclear microinjection
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47.  DNA coated on pellets (gold
particles) is forced down to the
barrel of a ‘Particle Gun’ by an
explosive charge.
 The particles are forced through
the cell wall where the DNA is
released.
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48.  The electrostatistically coated poly (beta-amino ester) nanoparticles can facilitate
ligand-mediated gene delivery.
 The more promising polymers for gene delivery is degradable poly(beta-amino
ester), 1,3-diaminopentane-terminated poly (5-amino-1-pentanol-co-1,4-butanediol
diacrylate) (C32-117).
 This polymer function 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 bio-distribution and lack of tissue-specific targeting
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51.  A short controlled pulse of electricity to cell momentarily disrupting lipid bilayer.
 Steps:-
 Use of high-voltage electric shocks to introduce DNA into cells.
 Voltage results in temporary breakdown and formation of pores.
 Small pores (40-120nm) reseal quickly.
Harvest cell and resuspend in electroporation buffer
Add DNA to cell suspension for stable transfection DNA should be lineralized, for
transient the DNA may be supercoiled
Electroporate
Selection process for transfectant
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57.  Liposomes are made up of phospholipid
bilayer which can carry both hydrophilic as
well as hydrophobic substances. Therefore,
commonly used in drug delivery.
 Lipofection (or transfection) is a technique
used to inject genetic material into a cell by
means of liposomes., which are vesicles that
can easily fuse with the cell membrane
(endocytosis).
 Advantages:
Safer when compared to viral vectors
Can carry large DNA molecules
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59.  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 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.
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62. Inherited Disease
A large number of disease are known to be inherited from the parents to the offsprings.
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: These disorders are caused by genes present on the sex chromosomes.
The abnormal disorders express more commonly in males than females.
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63. Disease Gene Therapy
Severe Combined Immunodeficiency (SCID) Retroviral transfer of enzyme Adenosine deaminase
gene into lymphocyte
Familial Hypercholestrolemia Retroviral transfer of LDL receptor gene into
hepatocytes
Hemophilia Retroviral transfer of gene of factor VIII & IX into
fibroblasts
Cystic Fibrosis CFTR gene transfer into bronchial epithelium by
Adenovirus
Duchenne Muscular Dystrophy Retroviral transfer of dystrophin gene
Leber’s Hereditary Optic Neuropathy Transfer of gene of the enzyme, isomer hydrolase,
by adenovirus into the retina
Lesch-Nyhan Syndrome Retroviral transfer of HGPRT into lymphocytes
Sickle Cell Anemia Transfer of beta globin gene by adenoviral vector
Thallasemia Transfer of α & β by an adenoviral vector
Gaucher’s Disease Transfer of β-glucosidase by liposome
Cancer (Wilm’s Tumor) Insertion of p53 tumor suppressor gene by liposome 63

64.  SCID is caused by deficiency of the enzyme Adenosine Deaminase (ADA)
– Gene is located on chromosome 22
– Deficiency results in failure to develop function B & T 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 levels of dATP produce a general deficiency of other dNTPS in T lymphocytes
– B cells doesn’t mature as they require help of T cells
– Patients cannot withstand infection  Die if untreated
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68.  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
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69. –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)
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71. Severe OTC deficiency
 Newborns  coma within 72 hours
 Most suffer severe brain damage
 ½ patients 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
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72. Inability to produce ornithine transcarbamylase (OTC) is often lethal, but moderate
deficiencies may be controlled by strict control of diet.
Jess Gelsinger, a young volunteer in a gene therapy trial who had a moderate OTC
deficiency, died on 17 Sept 1999
He had been injected in the liver with high concentrations of adenovirus that
expressed OTC
He apparently died of a massive immune response to the adenovirus vector
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74.  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 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 atherosclerotic disease resulting
in fatal cardiovascular events beginning in the second & third decades.
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75.  Class I: LDL receptor (LDLR) is not synthesized at all.
 Class II: LDLR is not properly transported from the endoplamic reticulum to the
Golgi apparatus for expression on the cell surface.
 Class III: LDLR does not properly bind LDL on the cell surface (this may be
caused by a defect in either Apolipoprotein B100 or a defect in LDLR.
 Class IV: LDLR bound to LDL does not properly cluster in clathrin-coated pits for
receptor-mediated endocytosis
 Class V: The LDLR is not recycled back to the cell surface
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77.  Major issue is LDL receptor mutation.
 For Familial hypercholesterolemia there are 806 mutations. Out of which 457
mutations are missense and nonsense.
 Substitution mutations
 GGG-AGG Gly-Arg Hypercholesterolemia
 GCG-GAGAla-Glu Hypercholesterolemia
 CTC-CCC Leu-Pro Hypercholesterolemia
 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
– Has been used in many trials since then
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79.  Cystic fibrosis (CF) is an inherited
autosomal recessive disorder
 CF affects the epithelial cells lining
air passage to the lungs
 CF affects the epithelial cells lining
air passages to the lungs
 CF causes a buildup of thick mucus
in the airways
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80.  In CF, there is a defective ion channel protein i.e., Cystic fibrosis
transmembrane conductance regulator (CFTR) protein
 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 ?
–Adenovirus vector was used to deliver a normal ion channel
protein to airway cells in a patient’s nose or lungs.
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82.  Thalassemia 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 β-
chains 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.
 Gene transfer of a regulated β-globin gene in Hemopoietic stem cells (HSCs)
would reduce the imbalance between α or 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.
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85.  It is an X-linked recessive disorder.
 It mostly affects male as they contain a single copy of X-chromosome.
 In females, a mutation must usually be present in both copies of the gene to cause
the disorder.
 There is deficiency of the enzyme Hypoxanthine-guanine phosphoribosyl
transferase (HGPRT) or Kelly Seegmiller syndrome that affects the levels of uric
acid in the body.
 Gene therapy involves retroviral transfer of HGPRT gene in lymphocytes.
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87. 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 genotype – homozygous.
Sickle cell trait – A mild condition where 50% of the red blood cells are affected. Sickle
cell trait individuals are heterozygous, having one of each allele
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88. Genotypes Phenotypes
HbNHbN Normal haemoglobin
HbNHbS Sickle cell trait
HbSHbS Sickle cell trait
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89. • Efficient gene transfer into target cells (Insertional mutagenesis)
• Adequate level of transgene expression
• Persistence of gene expression
• Regulation of gene expression
• Tolerance to transgene product
• Safety
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91.  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
̶ Patients 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.
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92. 1. Inefficient gene delivery – not suitable for all genetic diseases
2. Most effective if stem cells are involved
 Only to correct a few cells with the gene
 E.g. Blood stem cells: SCIDS and Gaucher’s Disease
3. Less effective or Ineffective if many cells must be corrected
 Brain cells (Tay-Sachs disease, Huntington’s disease)
 Cystic fibrosis
4. Insertion of gene is not always permanent
 E.g. Gaucher Disease: temporary cure until β-glucosidase gene “popped” out of
chromosome
5. Insertion of gene into genome could disrupt other genes
 Possible consequences?
6. Some viruses elicit immune response or may cause disease
 E.g. Jesse Gelsinger died in 1999
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