Gene

Therapy
 Gene Therapy
⚫ Defined as the use of genetic manipulation
for treatment genetic disorders

⚫ Other Definition:
The treatment of medical disorders by the delivery
of therapeutic genetic information into the
appropriate cellular targets

⚫ Two critical steps are required for gene therapy using

gene transfer techniques:

⚫ Appropriate transfer of gene(s) or genetic material

⚫ Continued gene expression at appropriate levels for
therapy
 Why gene therapy over drug therapy?
⚫ Can replace a dysfunctional gene or deficient gene.

⚫ Transgene can result into continuous production of a

therapeutic protein that normally has a short half life.

⚫ Can be focused to a specific cell type to avoid

potentially toxic systemic effects.

⚫ Can improve patient's compliance.

⚫ And decrease cost of therapy on long term bases

Main strategies for gene therapy

⚫ Gene addition

⚫ Removal of a harmful gene by antisense

nucleotide or ribozymes

⚫ Control of gene expression

 HISTORY AND DEVELOPMENT
OF GENE THERAPY
1990:
• The first approved gene therapy case at the National Institute
of Health, U.K. It was performed on a four year old girl named
Ashanti DaSilva. It was a treatment for a genetic defect that
left her with an immune system deficiency

• New gene therapy approach repairs errors in messenger RNA

derived from defective genes. This technique has the potential
to treat the blood disorder Thalassaemia, Cystic fibrosis, and
some cancers

• Sickle cell disease is successfully treated in mice

continued…….
1992
Doctor Claudio Bordignon-working at the Vita-Salute San Raffaele University,
Milan, Italy - performed the first procedure of gene therapy using hematopoietic
stem cells as vectors to deliver genes intended to correct hereditary diseases. This
was a world first.

1993
Andrew Gobea born wiith severe combined immunodeficiency (SCID). Blood was
removed from Andrew's placenta and umbilical cord immediately after birth,
containing stem cells. Retroviruses and stem cells were mixed, after which they
entered and inserted the gene into the stem cells' chromosomes.

1999
Death of Jesse Gelsinger in a gene-therapy experiment resulted in a significant
setback to gene therapy research in the United States

2002
Sickle Cell treated in mice by process of Gene Therapy

2003
A University of California research team inserted genes into the brain using
liposomes coated in a polymer called polyethylene glycol (PEG).
continued…….
2006
Scientists at the National Institutes of Health, successfully treated
metastatic melanoma in two patients using killer T cells genetically
retargeted to attack the cancer cells. This study constitutes one of the first
demonstrations that gene therapy can be effective in treating cancer.

May 2006
A team of scientists led by Dr. Luigi Naldin reported a breakthrough for
gene therapy in which they developed a way to prevent the immune
rejection using miRNA

1 May 2007
Moorfields Eye Hospital and University College London's Institute of
Ophthalmology announced the world's first gene therapy trial for inherited
retinal disease.

September 2009
The journal Nature reported that researchers at the University of
Washington and University of Florida were able to give trichromatic vision
to squirrel monkeys using gene therapy, a hopeful precursor to a treatment
for color blindness in humans
continued…….
2011:
Medical community accepted that it can cure HIV as in 2008, Gero
Hutter has cured a man from HIV using gene therapy

2017:
Research is still ongoing and the number of diseases that has been
treated successfully by gene therapy increases.
✔Adrenoleukodystrophy - The patients were children who had
inherited a mutated gene causing a rare disorder,
adrenoleukodystrophy, or ALD.
✔The disease strikes about one in 20,000 boys
✔The study involved 17 boys (the disease strikes males almost
exclusively), ages 4 to 13. All got gene therapy. Two years later, 15
were functioning normally without obvious symptoms.
 Chronology of main scientific steps
of Gene Therapy

Kratzer, K., Getz, L.J., Peterlini, T. et al. Addressing the dark matter of gene therapy: technical and ethical barriers to clinical application.Hum Genet 141, 1175–1193
(2022) https://doi.org/10.1007/s00439-021-02272-5
 The Beginning…
⚫The first human gene therapy proposed by Rogers
(1970) - that exogenous good DNA can be used to
replace the defective DNA in those who suffer from
genetic defects
⚫ In the 1980s, gene transfer methods to
mammalian cells were developed
◦ They would insert human genes into a bacterial cell.
◦ Then the bacteria cell would transcribe and translate
the information into a protein
◦ Then they would introduce the protein into
human cells
Cynthia and Ashanthi in 1992 with the pioneer physicians of gene therapy: (from left) French
Anderson, MD; Michael Blaese, MD; and Kenneth Culver. Four months later 10-year old
Cindy’s identical treatment followed.

R. Michael Blaese, MD with Ashanthi DeSilva (left) and Cindy Kisik at the IDF 2013 National
Conference, June 29.
Different approaches of Gene Therapy

Gene
Therapy

Somatic
Gene Germ line
therapy

Not much
In vivo Ex vivo actively
investigated
 Ex vivo gene therapy
• Cells from diseased person
are removed
• Then, they are treated in lab
(using techniques similar to
bacterial transformation)
• Finally, they are reintroduced
to the patient
• More effective than in vivo
• Transfection is the introduction of DNA into animal or plant
cells
Ex-vivo gene therapy
The First Case
⚫ The first gene therapy was performed on
September 14th, 1990

◦ Ashanti DeSilva was treated for SCID (Severe

combined immunodeficiency)
◦ Lack of functioning immune system because of
defect in gene called (ADA), adenosine deaminase
which is involved in metabolism of dATP
(nucleotide precursor used for DNA synthesis
◦ Doctors removed her white blood cells,
inserted the missing gene into the WBC, and then
put them back into her blood stream.
◦ This strengthened her immune system
 First gene
therapy trial
for ADA
deficiency
-1990
 In vivo gene therapy

• Introducing genes directly

into tissues or organs
without removing body cells

• Challenge is delivery only to

intended tissues

• Viruses act as vectors for

gene delivery, but some
injected directly into tissue
In Vivo Gene Therapy for
Cystic Fibrosis
 Clinical Features
• Cystic fibrosis is a heterogeneous recessive genetic disorder
with features that reflect mutations in the cystic fibrosis
transmembrane conductance regulator (CFTR) gene.

• 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.
• Cystic fibrosis is a heterogeneous recessive genetic disorder with
features that reflect mutations in the cystic fibrosis transmembrane
conductance regulator (CFTR) gene.
• Cystic fibrosis was first described as a disease in the late 1930s by
Dorothy Hansine Andersen. In 1988, the first mutation for CF, ΔF508,
was discovered by Francis Collins, Lap-Chee Tsui and John R.
Riordan on the 7th chromosome of the human genome

The mutation for CF on the 7th chromosome

• 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 non classic 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.
Molecular Genetics and Gene Function

Locus: 7q31.2 - The CFTR gene is found in region q31.2 on the long
(q) arm of human chromosome 7.
Gene Structure: The normal allelic variant for this gene is about
250,000 bp long and contains 27 exons.
mRNA: The intron-free mRNA transcript for the CFTR gene is 6129
bp long.
Coding Sequence (CDS): 4443 bp within the mRNA code for the
amino acid sequence of the gene's protein product.
Protein Size: The CFTR protein is 1480 amino acids long and has a
molecular weight of 168,173 Da.
Protein Function: The normal CFTR protein product is a chloride
channel protein found in membranes of cells that line passageways
of the lungs, liver, pancreas, intestines, reproductive tract, and skin.
CFTR is also involved in the regulation of other transport pathways.
Associated Disorders: Defective versions of this protein, caused by
CFTR gene mutations, can lead to the development of cystic
fibrosis (CF) and congenital bilateral aplasia of the vas deferens
(CBAVD).
 Protein Structure and Function
• CFTR transports chloride ions (Cl-) ions
across the membranes of cells in the lungs,
liver, pancreas, digestive tract,
reproductive tract, and skin.
• CFTR is made up of five domains:
• two membrane-spanning domains
(MSD1 and MSD2) that form the
chloride ion channel
• two nucleotide-binding domains
(NBD1 and NBD2) that bind and
hydrolyze ATP (adenosine
triphosphate)
• and a regulatory (R) domain.
• Delta F508, the most common CF-causing
mutation, occurs in the DNA sequence that
codes for the first nucleotide-binding
domain (NBD1).
3D Image of Protein
• When a CFTR protein with the delta F508
mutation reaches the ER, the quality-control
mechanism of this cellular component recognizes
that the protein is folded incorrectly and marks
the defective protein for degradation. As a result,
delta F508 never reaches the cell membrane.
• People who are homozygous for delta F508
mutation tend to have the most severe symptoms
of cystic fibrosis due to critical loss of chloride
ion transport.
• This upsets the sodium and chloride ion balance
needed to maintain the normal, thin mucus layer
that is easily removed by cilia lining the lungs
and other organs. The sodium and chloride ion
imbalance creates a thick, sticky mucus layer that
cannot be removed by cilia and traps bacteria,
resulting in chronic infections.
Hallmarks of CF

• Very salty-tasting skin

• Appetite, but poor
growth & weight gain
• Coughing, wheezing &
shortness of breath
• Lung infections, e.g.
pneumonia/bronchitis
Protein Function and Biochemistry

• Cystic Fibrosis
Transmembrane
conductance
Regulator (CFTR)
controls chloride ion
movement in and
out of the cell.
Protein Function Continued

As a result, mucociliary clearance and bacterial killing are

impaired making CF airways vulnerable for infection and inflammation.
 Presentation of Disease

Mucous in the airways cannot be easily cleared from the lungs.

 Presentation of Disease
Colon

Pancreas

Sticky mucus secretion

Ducts are filled with sticky mucus. Scaring of tissue

 Treatment
•
The only way to cure CF would be to use gene therapy to replace the defective gene or to give
the patient the normal form of the protein before symptoms cause permanent damage

•
The major goal in treating CF is to clear the abnormal and excess secretions and control
infections in the lungs, and to prevent obstruction in the intestines.

•
For patients with advanced stages of the disease, a lung transplant operation may be necessary.

•
Although treating the symptoms does not cure the disease, it can greatly improve the quality of
life for most patients and has, over the years, increased the average life span of CF patients to
30 years.

Gastrointestinal Treatment

Modified diet
Due to pancreatic disorders, children with CF require a modified diet, including vitamin
supplements (vitamins A, D, E, and K) and pancreatic enzymes. Maintaining adequate nutrition
is essential. The diet calls for a high-caloric content (twice what is considered normal for the
child's age), which is typically low in fat and high in protein. Patients or their caregivers should
consult with their health care providers to determine the most appropriate diet.
 Gene Therapy for Cystic
Fibrosis
Cystic fibrosis should be an ideal candidate for gene
therapy, for four main reasons:
•it is a single gene defect
•it is a recessive condition, with heterozygotes being
phenotypically normal (suggesting gene dosage
effects are not critical)
•the main pathology is in the lung, which is
accessible for treatment
•it is a progressive disease with a virtually normal
phenotype at birth, offering a therapeutic window.
• In the case of CF, gene
therapy involves
inhaling a spray that
delivers normal DNA
to the lungs.
• The goal is to replace
the defective CF gene
in the lungs to cure CF
or slow the progression
of the disease.
 History
• The discovery and cloning of the CFTR gene 1989
• Since 1993, 25 safety clinical trials have been carried
out.
• First trials mostly focused on the nasal epithelium then
directly into the lungs.
• Adenovirus vectors were first used, until it was clear that
there was no adenoviral receptor in airway epithelial
cells.
• Adenoviral vectors were replaced with adeno-associated
vectors, but the most recent ones were unsuccessful.
• Other 9 clinical trials have used nonviral GTA’s that are
more effective.
• Nebulizer spray
 Gene therapy for cystic fibrosis
Barriers : Circulating antibodies,
Mucus layer, Macrophages, Immune
response

Vectors : Viral- Adenovirus,

Adenovirus-Associated, Lentivirus,
Poxvirus, Herpesvirus
Nonviral- plasmid

Recent breakthrough: Nebulized

liposome-mediated gene therapy
 Nebulizer Spray
• Lung delivery of plasmid DNA encoding the CFTR gene complexed
with a liposome is a potential treatment option for patients with
cystic fibrosis.
 Delivery of therapeutic genes

•Therapeutic genes often

called payload
•May require long- term
expression of corrective
gene
•Others require rapid
expression for short
periods of time
Methods of Gene Transfer
The gene transfer methodologies that are clinically exploitable by
gene therapy can be divided into four categories.

⚫ Simple utilization of naked plasmids (circular,

covalently closed DNA molecules) or short
regulatory nucleic acids (oligonucleotides, siRNAs, and
others), not complexed with other molecules and simply
injected in vivo or added to the extracellular milieu of
cultured cells.
⚫ Facilitation of nucleic acid entry into the cells by physical
methods.
⚫ Transport of nucleic acids into the cells by lipofection.
⚫ Embedding of nucleic acid sequences within viral
genomes, then exploiting the natural property of viruses
to enter target cells at high efficiency.
 ⚫ RNA viruses (Retroviruses)
◦ Murine leukemia virus (MuLV)
◦ Human immunodeficiency viruses (HIV)
◦ Human T-cell lymphotropic viruses (HTLV)
vectors ⚫ DNA viruses
◦ Adenoviruses
◦ Adeno-associated viruses (AAV)
◦ Herpes simplex virus (HSV)
◦ Pox viruses
⚫ Non-viral vectors
◦ Liposomes
◦ Naked DNA
◦ Liposome-polycation complexes
◦ Peptide delivery systems
 Choices of Vectors
Viral vectors: Non-viral vectors:
Retrovirus Liposome
Adenovirus DNA–polymer conjugates
Naked DNA
Adeno-associated virus
Herpes Simplex Virus
The ideal vector system would have the following
characteristics:

•An adequate carrying capacity

•To be undetectable by the immune system
•To be non-inflammatory
•To have long duration of expression and/or the ability to be
safely re-administered
 The Ideal Vector for Gene Transfer
• High concentration of virus allowing many cells to be infected or
transduced
• Convenience and reproducibility of production

• Ability to transduce dividing and non-dividing cells

• Ability to integrate into a site-specific location in the host chromosome,

or to be successfully maintained as stable episome

• A transcriptional unit that can respond to manipulation of its regulatory

elements

• Ability to target the desired type of cell

• No components that elicit an immune response

 How to fix it
B C A a beneficial gene
A
Virus Modified virus

•A virus is found which replicates by inserting its genes into the host
cell's genome. This virus has three genes - A, B and C.

•Gene A encodes a protein which allows this virus to insert itself into
the host's genome.

•Genes B and C actually cause the disease this virus is associated with
Replace B and C with a beneficial gene. Thus, the modified virus
could introduce your 'good gene' into the host cell's genome without
causing any disease.

So we use the modified virus to fix the “broken window”

 Why use viral vectors?
• Virus are obligate intracellular parasites

• Very efficient at transferring viral DNA into host cells

• Viral vectors use viral genome to carry therapeutic

gene(s) and to infect human body cells

• Specific target cells: depending on the viral attachment

proteins (capsid or glycoproteins)

• Gene replacement: non-essential genes of virus are

deleted and exogenous genes are inserted

• Viruses must be engineered so that they can neither

produce disease nor spread (extremely effective at
infecting human cells)
 VIRUSES AS VECTORS
• Any virus can potentially be used to express foreign
genes

• Different viruses are better suited for different kinds of

uses

• Integration may be important, such as in many

gene therapy uses

• Larger viruses can express more and larger

foreign genes but are more difficult to manipulate

• The cis-acting promoters for genome replication and

packaging must be understood
 Potential Uses of Viral Vectors
• Gene therapy to replace a missing or inadequate
gene

• Cure of illness by expressing a reagent to, for

example, kill cancer cells

• Immunization by expressing an antigen from a

pathogen

• Expression of genes in cultured cells for scientific

study
 Introduction of Genes Into Animals
VIRAL VECTORS MAJOR LIMITATIONS

Papova (SV40, Polyoma) Size; Host range

Papilloma (BPV) Size; Integration,Transformation
Adeno associated (AAV) Size; production
Adeno Size; antigenicity, episomal DNA,
toxicity
Herpes/Vaccinia Pathogenic, cytotoxic, lytic
Retroviruses Inability to infect post-mitotic
cells
Lentiviruses Safety, integration
Introducti
on to virus
 History
Louis Pasteur was unable to find a causative agent for rabies and speculated about a
pathogen too small to be detected using a microscope.

In 1884, the French microbiologist Charles Chamberland invented a filter (known

today as the Chamberland filter or the Pasteur-Chamberland filter) with pores
smaller than bacteria. Thus, he could pass a solution containing bacteria through
the filter and completely remove them.

In 1892, the Russian biologist Dmitri Ivanovsky used this filter to study what is now
known as the tobacco mosaic virus. His experiments showed that crushed leaf
extracts from infected tobacco plants remain infectious after filtration. Ivanovsky
suggested the infection might be caused by a toxin produced by bacteria, but did
not pursue the idea.
In 1898, the Dutch microbiologist Martinus Beijerinck repeated the experiments and
became convinced that the filtered solution contained a new form of infectious
agent. He observed that the agent multiplied only in cells that were dividing, but
as his experiments did not show that it was made of particles, he called it a
contagium vivum fluidum (soluble living germ) and re-introduced the word virus.
 Definition of viruses

• Are infectious agents that are too small to be

seen with a light microscope .

• Acellular (absence of nucleus, organelles,

cytoplasm,plasma membrane).
• No ATP generating metabolism

• Do not undergo binary fission

• Sensitive to interferon
General Characteristics of Viruses

• Obligatory intracellular parasites

• Contain DNA or RNA
• Contain a protein coat
• Some are enclosed by an envelope
• Some viruses have spikes
• Most viruses infect only specific types of cells
in one host
• Host range is determined by specific host
attachment sites and cellular factors
 General characteristics

• Viruses replicate through replication of their

nucleic acid and synthesis of the viral protein.
• Viruses do not multiply in chemically defined
media
• Some viruses have enzymes inside the virion. All
ss- RNA viruses with negative polarity have the
enzyme transcriptase ( RNA dependent RNA
polymerase) inside virions.
• Retroviruses and hepatitis B virus contain the
enzyme reverse transcriptase.
 Components of viruses
• Nucleic Acid Core (DNA
or RNA)
• Capsid: Surrounding
protein coat
• Envelope: Some
viruses have this
additional surrounding
lipid bilayer membrane
•
Virion: A complete
virus particle
 Function
Viruses use their nucleic acids (genome) to replicate
themselves in host cells
Capsids also play a key role in the attachment of some viruses.
Each capsid is composed of protein subunits called
capsomeres.
Enveloped viruses have a typical bilayer membrane outside
their capsids and acquire their envelope after they are
assembled in a host cell and “bud” through host’s membrane.
Helps to protect from drying (enhances transmission),makes
virus more susceptible to chemical agents that dissolve
lipids and helps to attach to host cell membrane.
Nucleocapsid comprises the viral genome together with the
capsid
 Naked: viruses with a nucleocapsid and no
envelope

Spikes: projections that extend from the viral

envelope that may aid in attachment to the host cell

Glycoprotein: these surface projections serve to

attach virions to specific receptor sites on
susceptible host cell surfaces
Viral morphology
Virus Sizes and shapes
Viral
morphology
• Polyhedral viruses

• Enveloped viruses

• Helical viruses

• Complex viruses
Morphology of a Polyhedral
Virus
Polyhedral Viruses
Morphology of an Enveloped Virus
Figure 13.3
Enveloped Viruses
Morphology of a Helical Virus
Morphology of
a Complex
Virus
 Viral shape

•Helical capsid: consists of a ribbonlike protein that

forms a spiral around the nucleic acid
•Polyhedral capsid: many-sided, and one of the most
common polyhedral capsid shapes is the icosahedron
•Some viruses have a bullet-shaped capsid and some

are spherical
Viral genome
structure
 TERMINOLOGIES
• VIRION – a complete viral particle

-in naked viruses virion is identical to the nucleocapsid

-in enveloped viruses  must acquire envelope before it
is considered a virion
• NUCLEOPCAPSID – a protein-nucleic acid complex

• VIROIDS – consist solely of a single molecule of circular

RNA without a protein coat or envelope
• PRIONS – infectious protein particles composed solely of
proteins
 Host range and specificity of
viruses
• Most viruses infect specific host cells ie are host
specific. Host specificity is due to:
• specific attachment sites on the host cells called
receptors
• Receptor sites for bacteriophage are found in
bacterial cell walls or fimbrae or flagella
• Animal cell membranes contain receptors for animal
viruses availability of cellular factors required for
viral multiplication in the host cells.
 Retrovirus

• The term "retro" in retrovirus refers to the reversal of

the central dogma of molecular biology.

• A retrovirus is a single-stranded positive sense RNA

virus that replicate via a double-stranded DNA
intermediate.

• It is roughly 100 nanometers in diameter.

• It is one of the mainstays of current gene therapy

approaches.
 Retrovirus
• Enveloped virus with lipid bilayer and viral spike
glycoproteins.
• Have outer matrix protein and inner core capsid containing
viral genome.
• Genome: Two copies of single stranded positive-stranded
RNA (8-10kb).
• Reverse transcriptase to generate DNA
• Viral genes are integrated into host genome. Progeny virus
produced using host cell transcriptional and translational
machinery.
 Retroviruses as vectors

•Most retroviruses do not kill the host, but produce progeny

virions over an indefinite period.
•Retroviral vectors can therefore be used to make stably
transformed cell lines (undergo phenotypic change).

• Viral gene expression is driven by strong promoters,

which can be subverted to control the expression of
transgenes.

• Murine mammary tumor virus (Transcription)

 Inducible by glucocorticoids
Have a broad host range allowing the transduction of many
cell types.
•E.g. amphotropic strains of murine leukemia virus (MLV)

•Make efficient and convenient vectors for gene transfer

because the genome is small enough for DNA copies to be
manipulated in vitro in plasmid-cloning vectors..
Adenovirus:

• Nonenveloped particle
• Contains linear double
stranded DNA
• Does not integrate into
the host genome
• Replicates as an episomal
element in the nucleus
Adeno virus as drug delivery system

⚫ most use adenovirus 5;

infects dividing and
nondividing cells
⚫ can enter many different cell
types because it uses
⚫ a relatively ubiquitous
receptor for entry (CAR)
⚫ produces very high
titers of viral particles
⚫ Non-enveloped virus –
relatively stable virion
Drawbacks of using Adenovirus
⚫ transient expression - adenovirus is not stably maintained
in cells – expression is usually lost over a several
week period
⚫ adenoviruses are highly immunogenic - cannot
effectively introduce multiple rounds of the same
adenovirus serotype in vivo, but there are several different
serotypes of adenoviruses. Would need to put your gene of
interest into each different serotype if you wanted to
perform multiple transductions in vivo
⚫ can be pathogenic – 1999 University of Pennsylvania gene
therapy death using the largest quantity of Ad5 vector into
a human – severe immune response and coagulation with
no evidence of gene expression.
⚫ productive viral replication is highly cytolytic to
the infected cell, in vivo delivery system use defective
virus.
 Other Viral vectors
⚫ Adeno-associated virus – infects wide range
of host cells (both dividing and non-
dividing), able to integrate into genome,not
associated with any human disease,
high efficiency of transduction.
⚫ Herpes simplex virus, vaccinia virus,
syndbis virus
⚫ Onyx virus – limited replicating adenovirus
that replicates mainly in tumor cells
Adeno-associated viral vectors:

• AAV is a simple, non-pathogenic, single stranded DNA

virus dependent on the helper virus (usually adenovirus) to
replicate.

• It has two genes (cap and rep), sandwiched between

inverted terminal repeats that define the beginning and the
end of the virus and contain the packaging sequence.

• The cap gene encodes viral capsid proteins and the rep
gene product is involved in viral replication and integration.

• It can infect a variety of cell types and in the presence of

the rep gene product, the viral DNA can integrate
preferentially into human chromosome 19.
• To produce an AAV vector, the rep and cap genes are replaced with a
transgene.

• The total length of the insert cannot exceed 4.7 kb, the length of the wild
type genome.

• Production of the recombinant vector requires that rep and cap are
provided in trans along with the helper virus gene products.

• The current method is to cotransfect two plasmids, one for the vector and
another for rep and cap into cells infected with adenovirus.

• This method is cumbersome, low yielding and prone to contamination

with adenovirus and wild type AAV.

• Interest in AAV vectors is due to their integration into the host genome
allowing prolonged gene expression.
Adeno-associated virus vectors:
Advantages:
All viral genes removed
Safe
Transduction of nondividing cells
Stable expression
Disadvantages:
Small genome limits size of foreign DNA
Labor intensive production
Status of genome not fully elucidated
 Adenovirus associated virus
(AAV)
⚫ Advantages:
◦ not associated with disease
◦ can obtain high titered virus stocks (109- 1010/ml)
◦ Small genome that is easy to manipulate
◦ stable integration into the genome in chromosome
19
◦ infects both dividing and nondividing cells
⚫ Disadvantages:
◦ can only contain ~4.5 kb of DNA
Lentiviral Vectors:
• Belong to the retrovirus family but can infect both dividing
and non-dividing cells.

• They are more complicated than retroviruses, containing an

additional six proteins, tat, rev, vpr, vpu, nef and vif.

• Human immunodeficiency virus (HIV) has been disabled

and developed as a vector for in vivo gene delivery.

• Low cellular immune response, thus good possibility for in

vivo gene delivery with sustained expression over six months.

• No potent antibody response.

 Alternative delivery
•Liposomes are small diameter, hollow particles made of lipid
molecules, packaged with genes and injected
into tissues

•A gene gun could also be used

•“Naked” DNA injected directly into body

tissues (ex. effective in liver/muscle), but
not enough cells express gene to have
affect
•Artificial chromosomes may also deliver therapeutic gene
•Non-protein coding DNA with therapeutic gene
•Similar construction to normal chromosomes (designed for
permanent incorporation)
Non-viral cancer gene therapy: Beyond delivery
 Physical Methods

⚫ Electroporation
⚫ Hydrodynamic Intravascular Injection
⚫ Sonoporation
⚫ Bombardment with DNA-Coated
Microparticles (“Gene Gun”)
⚫ Injection of DNA using High-Pressure
Jets
(“Jet Injection”)
 Chemical Methods
⚫ Liposomes and Cationic
Lipids
(Lipofection)
⚫ Cationic Polymers
⚫ Proteins
 Germline gene therapy

The desired gene is introduced in the germ

cell. The gene gets transferred from one
generation to another generation.

One can create designer babies

 Germline gene therapy
• The germline gene therapy targets germinal or reproductive
cells. These cells produce male and female gametes therefore
the inserted gene passes to the future generations.
• The transfer can also be done during early embryonic
development, e.g. during in-vitro fertilization, then the desired
gene can be inserted in all the cells of a developing embryo.
• The germline gene therapy can be more effective and can be a
permanent cure for genetic diseases that run in families. It has
the potential to eliminate a disease from the population.
• But it is not yet legal in many countries due to ethical issues.
Some people may use it for enhancements rather than
treatments. There is also insufficient knowledge of risks posed
to the future generations.
 Germline Gene Therapy
Limitations
Germline gene therapy is altering the organisms genes
before development into a fetus, however this illegal
in humans because they say:

• New human disease could be created

• Human evolution would be interfered
• Too many moral and ethical issues with changing a
human genome
• Potential negative effects on future generation