Gene therapy

Gene Therapy
Guided by
Dr. Manju Misra
M.Pharm, Ph.D.
Associate professor
GSP-GTU
Prepared by
Harshil S Jani
M.Pharm
Semester 2
202880820004

Flow of Presentation
Introduction
What is Gene therapy and Why we need Gene Therapy
Gene Therapy mechanism
Strategies for Gene Therapy
Types of Gene therapy
Characteristic required for effective Gene Therapy
Obstacle for Gene Therapy
GRADUATE SCHOOL OF PHARMACY- GTU 2

Introduction
• Each of us carries about half a dozen of defective genes.
• About one in ten people has, or will develop at some stage or later, an inherited genetic disorder and
approximately 2,800 specific conditions are known to be caused by defects(mutation) in just one of
patient's genes.
• Cystic fibrosis is found in one out of every 2,500 babies born in the western world
Presently, gene therapies for following diseases are being developed:
• Cystic fibrosis
• HIV infection
• malignant melanoma
• kidney cancer
• Gaucher’s Disease
• Breast cancer
• Lung cancer

What is Gene Therapy? And why it is used?
• Gene Therapy is a simple therapeutic
method depending on either replacing a
distorted gene by healthy one, or completing
a missing gene in order to express the
required protein (zhang et el., 2004)
• Gene Therapy is the insertion, alteration or
removal of genes within an individual’s cells
and biological tissues to treat disease.
• The most common form of gene therapy
involves the insertion of functional genes
into an unspecified genomic location in
order to replace a mutated gene, but other
forms involve directly correcting the
mutation or modifying normal gene.
• GT is used to correct a deficient phenotype
so that sufficient amounts of a normal gene
product are synthesized for improvement in
genetic disorder

Gene Therapy

Gene Therapy mechanism

Strategies for Gene Therapy
Corrective
Therapy
Correction of genetic
defects in target cells
Treatment of disease with
single gene disorders like
cystic fibrosis, hemophilia,
sickle cell anemia, β-
thalassemia, muscular
dystrophy
Cytotoxic
Gene Therapy
Destruction of target cells
using a cytotoxic pathway
Treatment of malignant
tumors, including ovarian,
breast and endometrial
carcinoma

Types of Gene Therapy

Types of Somatic Gene Therapy

Somatic Gene therapy

• In somatic gene therapy, the therapeutic genes are transferred into the somatic
cells.
• Somatic cells are non-reproductive. This therapy is viewed as more safer,
conservative approach because it affects only the targeted cells in patient.
• The inserted therapeutic genes will not pass on to the next generation so the
patient’s offspring still have a chance of contracting the diseases.
• Somatic gene therapy is effective in treating various kinds of diseases such as cystic
fibrosis, muscular dystrophy, cancer and certain infectious diseases.
• However, the effect of somatic gene therapy is usually short-lived. Because the
cells of most tissues ultimately die and are replaced by new cells, repeated
treatments over the course of the individual’s life span are required to maintain the
therapeutic effect.

Ex vivo Gene Therapy

• Ex vivo gene therapy involve the genetic alterations of cells, normally by using the
viral vectors and transplanting back to patient body in order to correct disorders.
• Ex vivo gene therapy modifies the cells outside the body and transplanted back
after selection and amplification.
• Ex vivo gene therapy normally target on bone marrow stem cells, liver cells, blood
vessel smooth muscle cells and tumor-infiltrating lymphocytes for cancer
treatment.
• The relative advantages of ex vivo gene delivery include its ability to target
selectively specific cell types for production of gene of interest before engrafting
the cells into host.
• Furthermore, it is immuno-compatible because cells are collected from patient to
avoid rejection.
• The potential disadvantages of ex vivo gene therapy include it has to be maintained
and genetically modified in vitro, host cells must be capable of dividing, thus certain
postmitotic cell populations such as neurons cannot be targets of transduction for
ex vivo gene therapy.
• Moreover, it is also invasive.

In vivo Gene Therapy

• In vivo gene therapy involves the direct introduction of the genetic materials into
the human body.
• Physical methods applied for in vivo gene therapy are based on making transient
penetration in cell membrane by mechanical, electrical, ultrasonic, hydrodynamic,
or laser based energy so that DNA entrance into the target cells is facilitated.
• The target tissues of this technique includes skin, lung, colon, muscle, pancreas,
liver, bone marrow, spleen and brain.
• The advantages of in vivo gene therapy include simplicity. Gene delivery is
accomplished by the single step of direct vector injection into the desired target
organ to correct the disorder.
• Furthermore, this technique has minimal invasiveness. This is due to procedures
involved in this technique is simple and safe.
• Not only that, it is repeatable. The location can be injected more than once using
in vivo gene delivery approaches.
• The potential disadvantage of this therapy include the non specificity of target cell
infection. Various different types of cells can be infected when in vivo vectors are
injected in CNS, including neurons, glia, and vascular cells.

Germ line Gene Therapy

• Germ line gene therapy involves the reproductive cells.
• In this therapy, the germ cells(sperms and eggs) are modified by the introduction of
functional genes into their genomes.
• Gene therapy using germ cells results in permanent changes that will be passed on
to the next generations,
• If done early in the embryonic development, such as during pre implantation
diagnosis and in vitro fertilization, the gene transfer can occur in all the cells of the
developing embryo.
• This offer the possibility of permanent removing of an inherited disorder from a
family line.
• However, this raises controversy and it is unethical to conduct in human. Germline
gene therapy which involve the alterations in germ cells and will be passed to the
offspring is not allowed.

Cancer Gene therapy
Clinical targets for gene therapy (Edelstein et al., 2007)
• Since the first human gene therapy trial
performed in 1989 by Rosenberg and his team,
who tried to treat advanced melanoma in five
patients (Rosenberg et al., 1990)
• More than 1500 clinical gene therapy trials
were performed up to 2010 (Herzog et al.,
2010)
• More than 66% of them were to treat cancer
while 9.1% of them were devoted to
cardiovascular diseases, and only 8.3% were
targeted at treating monogenic genetic
disorders (Edelstein et al.,2007)

• The tumour cells release
anti apoptotic proteins like
bcl 2 and thus overcome
the apoptosis process
• The levels of bcl 2 protein
keep on increasing and the
levels of Bax protein
decrease
• Similarly the caspase 3 and
caspase 9 activation is
reduced

Characteristics required for effective
gene therapy

Obstacles for Gene Therapy

Aptamers and Antisense Oligonucleotide
Drugs of future

Aptamers
• Aptamers are short single/double stranded
NAs that specifically bind (lock and key
mechanism) to proteins, other NAs, or
even small molecules
• Aptamers can be designed to interfere
with functioning of “harmful” proteins,
either directly (bind to protein) or
indirectly (bind to mRNA/DNA)
• They are similar to monoclonal antibodies
• Provide opportunities for structure-based
drug design strategies relevant to
therapeutic intervention
• Can be prepared using SELEX(Systematic
Evolution Ligands by Exponential
Enrichment )

Properties of Aptamers
• RNA/DNA or Protein oligonucleotide molecules
• Ability to bind to proteins with high affinity and specificity
• Adopt wash number of 3-D shapes- which enables them to bind tightly to specific molecular target
• Usually very small in size but can easily distinguish between very closely related protein also
• Highest specificity and affinity to the target
• Small size and similarity to endogenous molecules
• Ability to disrupt interaction between proteins
• High specificity and high affinity to the target and are capable of penetrating tissues then antibodies because of their
smaller size
• Low or non immunogenic and non toxic molecules
• Production of aptamers is done in-vitro using automated synthesis
• Rationally design duration of action
• Less cost of oligonucleotide synthesis
• Function at different temperature, pH and buffers
• Unlimited shelf life

Difference between aptamer and antibodies

Aptamer
Drug
therapeutics
Drug delivery
systems
Bio
Imaging
In Western
Blot analysis
Diagnostic
tool

Aptamer as drug therapeutics
• Due to specific and tight affinity to target molecules, and low or no immunogenicity and toxicity, aptamers
are expected to be effective therapeutics reagents
• The inhibitory aptamer can block the function of a pathological target
• Pegaptanib (Macugen®) is a USFDA approved antiangiogenic RNA aptamer that prevents blood vessel growth
by directly binding to intracellular VEGF (isoform of vascular endothelial growth factor ( that regulates
vascular permeability Pegaptanib is therefore useful in the treatment of wet age related macular
degeneration (wet AMD)
• Huang et al. selected a DNA aptamer (AptCTLA-4) and showed high affinity to cytotoxic T lymphocyte
antigen-4 (CTLA-4). AptCTLA-4 remained nearly intact after incubation in serum for 24 hours, demonstrating
relatively stable in serum. As a consequence, AptCTLA-4 inhibited both tumor cell proliferation and tumor
growth in animal models
• Programmed cell death protein 1 (PD-1) is an important immune-suppressive molecule of T cell receptor
signaling, and its ligand PD-L1 is an important target of cancer cells. The PD-1/PD-L1 axis is a critical pathway
that involves tumor immune evasion. Lai et al. identified a PD-L1 targeting aptamer and found that aptPD-L1
blocks the binding between PD-1 and PD-L1, and assists the T-cell function restoration, thus promoting the
lymphocyte proliferation and inhibition of tumor growth

• Subramanian et al. reported the overexpression of nucleolin in retinoblastoma cells. By investigating the
effect of aptamer especially targeting to nucleolin on the proliferation of RB cells, they demonstrated that
the proliferation of retinoblastoma cells was significantly inhibited
• Using aptamer to directly block the function of target molecules would be an attractive way for disease
therapy.
• However, it would be difficult to find an aptamer with the antagonistic effects other than with only binding
affinity

Aptamer as Drug delivery sysytem
Aptamer Drug conjugates
• In this delivery system, the aptamer is directly linked (including
physical intercalating or covalent linking) with drugs or therapeutic
oligonucleotides.
• In the physical intercalation model, drugs containing an aromatic
nucleus, like Doxorubicin (DOX), daunorubicin, and Epirubicin (EPI),
can be simply loaded to the DNA double stands by physical
intercalating due to the π-π stack between the aromatic nucleus of
drugs and DNA base pairs. The intercalating is usually G-C base pairs
preferential.
• Yu et al. developed a HepG2-specific aptamer and modified it with
repeated G-C bases at 5'-end and intercalated with DOX .
• This system could achieve the specifically targeted delivery of DOX
(one molecule could be loaded with 4 molecules of DOX) into HepG2
cells but not normal cells, causing apoptosis and dose-dependent
cytotoxicity on HepG2 cells.

• The drug-aptamer conjugate can be formed by a
covalent bond In this approach, drugs can be either
directly linked to an aptamer or indirectly linked with
a spacer between them.
• Cytotoxic drugs, including gemcitabine, 5-fluorouracil
(5-FU), derivative of maytansine 1 (DM1) and
monomethyl auristatin E (MMAE) were conjugated
with the pancreatic cancer RNA aptamer P19 [34]
selected by Yoon et al., there are carbon linkers
between the drug and 5’ end of a sticky sequence.
• P10 conjugated with gemcitabine or 5-F-20-UTP
monophosphate showed an inducement to the
phosphorylation of histone and the inhibition of cell
proliferation in pancreatic epithelial carcinoma cells
(PANC-1 cells) and in gemcitabine-resistant pancreatic
cancer cell line.

Aptamer-Drug-Nanomaterial systems
• The combination of aptamer and nanomaterials, including liposome, gold nanoparticles, organic polymer,
dendrimer, mesoporous silica nanoparticles and carbon nanomaterials, has been proposed and provided
targeted therapy with expanded treatment methods.
Gold Nanoparticle
• Gold Nanoparticles (GNPs) have attracted great attention due to their favorable properties
such as surface plasmon resonance, fluorescence characteristics, supramolecular and
molecular recognition properties, photothermal effect and low-toxicity, stability, small size and
functionalizable surfaces that can be modified with different ligands
• An aptamer can be covalently conjugated to the GNPs through terminal sulfhydryl group. A
simple, efficient, and versatile system based on gold nanoparticle-DNA aptamer conjugate
(GNP-Apt) was developed by Ryou and his partners to deliver proteins into cells in vitro
without cytotoxicity
• Danesh et al. prepared a sgc8c aptamer modified and daunorubicin loaded gold nanoparticles
complex for the treatment of human acute lymphoblastic leukemia T-cell (Molt-4 cells).
Daunorubicin was intercalated in sgc8c aptamer and absorbed by GNPs via electrostatic
interaction between the negatively charged GNPs and positively charged daunorubicin (Fig.
6a). The complex could be more-efficiently internalized into Molt-4 cells and was less
cytotoxicity to non-targeted cells, compared with both single daunorubicin and even aptamer-
daunorubicin conjugate

Aptamer as Bio Imaging
• Another application is bio-imaging, using an aptamer that is conjugated to a fluorophore, or other materials
such as gadolinium, which is useful for magnetic resonance imaging (MRI).
• Using aptamers as imaging agents has the advantage of their being non-toxic, because oligonucleotide
moieties are present in the human body.
• As aptamers have high specificity for their target, accurate targeting, and rapid diffusion through the blood
circulation, use of these molecules can increase the certainty of the results obtained during diagnosis or
clinical analysis.
• Based on these advantages, aptamers have been studied as imaging agents for cell imaging as well as single-
protein imaging

Aptamers in western blot analysis
• Hah’s group published a new aptamer-based Western blot strategy that has reduced the procedure to one
step, and easily detects the target protein using only one aptamer
• Instead of two types of antibodies, the QD-conjugated RNA aptamer specific for the His-tag was employed.
This method has the advantages of requiring less time, not requiring antibodies or 32P, and introducing the
possibility of multiplexing detection
Aptamers as Diagnostic tool
• Aptamer based detection assays are expected to detect low concentration pathogens than
conventional antibody based detection assay such as ELISA
• Eg. HIV Strain R5 and Hepatitis B Virus

Aptamers
Advantages
• Produced chemically in a readily scalable
process
• Not prone to viral or bacterial contamination
• Non-immunogenic
• more efficient entry into biological
compartments
• Able to select for and against specific targets
and to select against cell-surface targets
• Can usually be reversibly denatured
• Dyes or functional groups can be readily
introduced during synthesis
Disadvantages
• Pharmacokinetic and other systemic
properties are variable and often hard to
predict
• Susceptible to renal filtration
• Have a shorter half-life
• Unmodified aptamers are highly susceptible
to serum degradation

Antisense Oligonucleotide
• These are short double stranded DNA/RNA
molecules that complimentarily bind to a specific
mRNA, release RNase H enzymes, thereby causing
mRNA hydrolysis and interference in the
translation process Accordingly, antisense
oligonucleotides have been successfully
employed to inhibit production of “disease
causing"proteins and cure conditions such as
hypercholesterolemia, neuromuscular disorders
and inflammatory disorders

• Alicaforsen is an FDA approved antisense oligonucleotide for the treatment of pouchitis
• Pouchitis is an inflammation of the surgically constructed internal pouch created in ulcerative colitis patients
who have had their diseased colons removed Alicaforsen prevents the production of intercellular adhesion
molecule 1 or ICAM 1 protein overexpressed in pouchitis
• Another antisense drug Mipomersen in Phase III clinical trials, acts against apo B protein to treat severe LDL
hypercholesterolemia

FDA Approved Antisense oligonucleotide

Concluding remarks
• Gene Therapy is promising approach in treatment of life threatening diseases such as cancer,
neurogenerative disorders and various inherited disorders
• Also, aptamer guided drug delivery as targeted drug delivery shows tremendous potential for site specific as
well tumor specific drug delivery
• Antisense oligonucleotide can be very useful for inhibition of production of disease causing peptide
• Aptamers and antisense oligonucleotide are the future of medicines

References
• Ibraheem, D., Elaissari, A., & Fessi, H. (2014). Gene therapy and DNA delivery systems. International Journal
of Pharmaceutics, 459(1–2), 70–83. https://doi.org/10.1016/j.ijpharm.2013.11.041
• He, F., Wen, N., Xiao, D., Yan, J., Xiong, H., Cai, S., Liu, Z., & Liu, Y. (2020). Aptamer-Based Targeted Drug
Delivery Systems: Current Potential and Challenges. Current Medicinal Chemistry, 27(13).
https://doi.org/10.2174/0929867325666181008142831
• Stein, C. A., & Castanotto, D. (2017). FDA-Approved Oligonucleotide Therapies in 2017. Molecular Therapy,
25(5), 1069–1075. https://doi.org/10.1016/j.ymthe.2017.03.023

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