its a presentation on gene therapy and its more emphasised on vectors of gene therapy.

1. PRESENTED BY
SUBODH S SATHEESH
MPHARM
PHARMACEUTICS
ECPS
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2.  Experimental technique for correcting defective genes that are
responsible for disease development
 The most common form of gene therapy involves inserting a
normal gene to replace an abnormal gene
 Other approaches used:
 Replacing a mutated gene that causes disease with a healthy
copy of the gene.
 Inactivating, or “knocking out,” a mutated gene that is
functioning improperly.
 Introducing a new gene into the body to help fight a disease
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3.  Each human being carries normal as well as some
defective genes
 In case of cystic fibrosis the mutant gene causes the
development of cysts and fibrous tissue in the patient’s
pancreas
 If the defective gene, however, is dominant, the disease
is expressed in any people that carry the defective gene
 Diseases such as heart disease do have a genetic
component, but are largely dependent on diet and
lifestyle.
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4.  Severe combined immuno-deficiencies (SCID)
 Hemophilia
 Parkinson's disease
 Cancer
 HIV
 Brain tomour
 Lung cancer
 Prostate cancer
 asthma
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6.  It involves the introduction of corrective genes to
reproductive cells or zygotes.
 It offers possibility for true care for several diseases.
 Result in permanent changes.
 Potential for offering a permanent therapeutic effect for
all who inherit the target gene. Possibility of
eliminating some diseases from a particular family.
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7.  Affects only the targeted cells in the patient, and is not
passed to future generations.
 Short-lived because the cells of most tissues ultimately
die and are replaced by new cells.
 Transporting the gene to the target cells or tissue is also
problematic.
 Appropriate and acceptable for many disorders,
including cystic fibrosis, muscular dystrophy, cancer,
and certain infectious diseases.
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9.  Gene augmentation therapy
 Targeted killing of specific cells
 Targeted inhibition of gene expression
 Targeted gene mutation
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12.  Genes are transferred to the cells grown in culture,
transformed cells are selected, multiplied and then
introduced into the patient.
 The use of autologous cells avoids immune system
rejection of the introduced cells.
 The cells are sourced initially from the patient to be
treated and grown in culture before being reintroduced
into the same individual.
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14.  It involves the transfer of cloned genes directly into the tissues of
the patient.
 Its done in case of tissues whose individual cells cannot be
cultured in vitro in sufficient numbers (like brain cells) and/or
where re-implantation of the cultured cells in the patient is not
efficient.
 Liposomes and certain viral vectors are employed for this
purpose because of lack of any other mode of selection.
 In case of viral vectors such type of cultured cells were often
used which have been infected with the recombinant retrovirus in
vitro to produce modified viral vectors regularly.
 The efficiency of gene transfer and expression determines the
success of this approach.
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16.  Plasmids or viruses used to move recombinant DNA from one
cell to other
 Adenoviruses retroviruses and liposomes are commonly used
 Ability to transfer and integrate genes into new cells
 Classified to
 viral
 Non viral
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17.  Retroviruses
 Adenoviruses
 Liposomes
 Herpes simplex virus
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19.  First used ones
 Retroviruses are diploid, single-stranded, circular-
enveloped RNA viruses of the family Retroviridae, with a
genome of 7–11 kb, and a diameter of approximately 80–
120 nm
 Retroviruses cause diseases such as AIDS, leukemia, and
cancer
 Retroviruses are viruses that integrate with host genome
to produce viral proteins (gag, pol, env) that are extracted
during gene delivery.
 Commonly used retroviruses are the Moloney murine
leukemia virus species, which have the capacity to deliver
exogenous genetic material up to approximately 9 kb
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21.  Viruses with double stranded DNA
 They can infect a broader variety of cells than
retroviruses
 It can cause respiratory, intestinal and eye infection
 adenoviruses Ad2 and Ad5 are The most widely studied
adenoviruses
 Penton and fiber proteins of virus capsid interact with
the coxsackievirus-adenovirus receptor cell surface
protein to provide cell binding
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22.  Lentivirals are viral systems without small, retrovirus-like
viral proteins and no capacity for replication
 The most important advantage of lentiviruses compared with
other retroviruses is their ability for gene transfer to non-
dividing cells
 Genome of lentiviruses have a more complicated structure;
they contain accessory genes which regulate viral gene
expression.
 HIV-1 is one of the most widely used lentiviral vectors, and
contains six accessory genes (tat, rev, vif, vpr, nef, vpu).
 Lentiviral vectors do not require degradation of the nuclear
membrane for integration
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23.  Nuerotropic virus
 Gene transfer to nervous system
 Can infect a wide range of tissues
 Complications are rare
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24.  Many studies that used viral vectors reported
unsatisfactory results, due to the immunologic and
oncogenic adverse effects of these vectors.
 It is overcome by NVGDS
 non-viral vectors have many advantages, such as easy of
fabrication, cell/tissue targeting, and low immune
response
 biggest disadvantage of non-viral vectors in clinical use is
low transduction efficiency.
 the biggest difficulty in gene therapy is the development
of physical methods to ensure gene transfer to target cells
of the gene delivery vectors and delivered gene.
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25.  Physical methods
 Chemical methods
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26.  Encapsulation
 Lipoplexes
 Dendrimers
 polyplexes
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27.  Gene guns
 Electroporation
 Ultrasound
 polymers
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28.  Delivery with gene gun method is also termed ballistic
DNA delivery or DNA-coated particle bombardment, and
was first used for gene transfer to plants in 1987.
 This method is based on the principle of delivery of
DNA-coated heavy metal particles by crossing them from
target tissue at a certain speed
 Generally, gold, tungsten or silver microparticles were
used as the gene carrier
 Gene-gun-based gene transfer is a widely tested method
for intramuscular, intradermal and intratumoral genetic
immunization.
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29.  Ultrasound has many clinical advantages as a gene delivery
system, due its easy and reliable procedure
 Microbubbles or ultrasound contrast agents decrease cavitation
threshold with ultrasound energy.
 Mostly perfluoropropane-loaded albumin microbubbles were
used.
 The transfection efficiency of this system is based on
frequency, time of ultrasound treatment, the plasmid DNA
mount used, etc
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30.  Electroporation includes controlled electric application to
increase cell permeability
 Electroporation introduces foreign genes into the cell by
electric pulses. In this method, pores are formed on the
membrane surface to enable the DNA to enter the cell.
 If the molecule is smaller than the pore size , it can be
transferred to the cell cytosol through diffusion
 loaded molecules and ions can be transported from the
membrane via electrophoretic and electro-osmotic means via
the effect of electric regions
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31.  Polymers are long-chained structures composed of small spliced
molecules called monomers.
 Polymers that are composed of a repeated monomer are called
homopolymers, while those composed of two monomers are called
copolymers.
 Biodegradable polymers are non-water soluble, and undergo
chemical or physical change in biologic environment.
 Polyamides, dextran, and chitosan are examples of biodegradable
polymers
 non-biodegradable polymers are not degraded in biological
environments;
 hydrophilic polymers are hydrogels, which are non-water soluble
and swell in water, while hydrophobic polymers are non-water
soluble and do not swell
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32.  CHEMICAL METHODS
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33.  An alternative to electrostatic condensation of DNA is
encapsulation of DNA with a biodegradable polymer.
 Polymers that have an ester linkage in their structures
(like polyesters) are hydrolytically degraded to short
oligomeric and monomeric compounds, which are more
easily discharged from the body.
 The degradation mechanism and DNA release can be
controlled by changing the physicochemical
characteristics and composition of the polymer.
 DNA is protected from enzymatic degradation by
encapsulation.
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34.  Deactivate the genes involved in the disease process
 Uses antisense specific to the target gene to disrupt the
transcription of the faulty gene
 Double stranded oligodeoxynucleotides as a decoy for
the transcription factors that are required to activate the
transcription of the target gene
 The oligonucleotide is designed to anneal with
complementarity to the target gene with the exception
of a central base, the target base, which serves as the
template base for repair.
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35.  Highly branched macromolecule with spherical shape
 Cationic dentrimers associate with nucleic acid
 the lack of ability to transfect some cell types, the lack of
robust active targeting capabilities, incompatibility with
animal models, and toxicity of cationic lipids are absent in
dendrimers
 Dendrimers offer robust covalent construction and extreme
control over molecule structure, and therefore size
 Producing dendrimers has been aslow and expensive process
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36.  Anionic and neutral lipids were used for the construction of
lipoplexes for synthetic vectors
 Cationic lipids, due to their positive charge, were first used to
condense negatively charged DNA molecules so as to facilitate
the encapsulation of DNA into liposomes
 Helper lipids (usually electroneutral lipids, such as DOPE)
were added to form lipoplexes, much higher transfection
efficiency was observed
 The most common use of lipoplexes has been in gene transfer
into cancer cells, where the supplied genes have activated
tumor suppressor control genes in the cell and decrease the
activity of oncogenes
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37.  Complexes of polymers with DNA are called polyplexes.
 polyplexes cannot directly release their DNA load into the
cytoplasm.
 low toxicity, high loading capacity, and ease of fabrication,
polycationic nanocarriers.
 co-transfection with endosome-lytic agents such as
inactivated adenovirus must occur.
 Use of polymers and copolymers help in the ease of
controlling the size, shape, surface chemistry of these
polymeric nano-carriers gives them an edge in targeting
capability and enhanced permeability.
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38.  To decide what is normal and what is disability
 To decide whether somatic gene therapy is more or less ethical
than germ line therapy
 Will the therapy only benefit the wealthy due to its high cost?
 Could the widespread use of gene therapy make the society
less accepting of people who are different?
 Should people be allowed to use gene therapy to enhance basic
human traits such as height, intelligence, or athletic ability?
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39.  Give a chance of a normal life to baby born with
genetic disease.
 Give hope of healthy life to cancer patient.
 For certain disease that do not have any cure except
gene therapy, it could save many lives.
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40.  The genetic testing, screening and research in finding the
availability of certain gene is very controversy.
 May increase rate of abortion if prenatal test regarding baby
with genetic disease is done.
 The cost is very high and the patient might need an insurance
to cover the treatment.
 Cosmetic industry may monopolized this gene therapy if it is
used in enhancing beauty and in vanishing the aging effect,
rather than used for treatment of a disease.
 Problems with viral vectors
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41.  Remington The science and practice of pharmacy
volume 1
 Nejm Gene therapy and novel drug delivery page 1-36
 Intechopem Gene therapy and viral and nonviral
vectors 387-402
 Japi.org Human gene therapy 1-17
 Anderson Germ line therapy spring 2013 1-25
 Nptel.ac.in gene therapy page 1-24
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