DNA

TEACHNOLOGY
And
Ethical issues
 Mat – general science
SCI. 408
SELECTED
TOPICS IN
HARLEQUIN RUTH
BIOLOGY
DR. JUDY E.
A. LAUSA
STUDENT GIGARE
PROFESSOR
TABLE OF CONTENTS

01 02

Gene therapy cloning

Introduction Introduction
Types Types
Methods Methods
Applications Applications
Risks Pros and Cons
Ethical issues Ethical issues
Gene therapy
INTRODUCTION
● Gene therapy involves altering the genes
inside your body's cells in an effort to
treat or stop disease.

● Gene therapy replaces a faulty gene or

adds a new gene in an attempt to cure
disease or improve your body's ability to
fight disease. Gene therapy holds
promise for treating a wide range of
diseases, such as cancer, cystic fibrosis,
heart disease, diabetes, hemophilia and
AIDS.
 BACKGROUND
Gene therapy has evolved over the years. Here are some of the most important milestones that have
brought us to where we are with gene therapy today: 

1953 The structure of DNA was characterized by a double helix

James Watson, Francis Crick, Maurice Wilkins, and
Rosalind Franklin
King’s College London
The genetic code was discovered by deciphering the three bases of DNA in 1 of
1961- the 20 amino acids. The 19 remaining amino acids were deciphered soon after,
1966 paving the way for new technologies
Marshall Nirenberg, Har Khorana, and Severo Ochoa
National Institutes of Health (NIH)
Researchers discovered a genetic engineering technique that allows genetic
1973 material from 1 organism to be artificially introduced, replicated, and
expressed in another
Stanley N. Cohen and Herbert W. Boyer
US-Japan joint meeting on plasmids, Hawaii
 One of the first times gene therapy was tested in people was done
without permission from the university who provided funding for the
1980 National Institutes of Health (NIH). The researcher lost multiple grants
and NIH warned others that human experimentation would not be
tolerated
Martin Cline
The University of California, Los Angeles

The first gene therapy clinical trial was conducted using new viral vector
1990 technology
Michael Blaese and French Anderson
National Institutes of Health (NIH)

The first engineered nuclease technology (zinc finger nuclease) was

1996 studied, laying the groundwork for exploring the use of zinc finger
nucleases for gene editing as a potential for gene therapy

Yang-Gyum Kim, Jooyuen Cha, and Srinivasan

Chandrasegaran
Johns Hopkins University
 The first generation of lentiviral vectors (LVVs) was created using 3 different
plasmids (a DNA structure that can replicate without a chromosome)
1996 containing a large deactivated portion of the HIV genome, making it unlikely
for HIV to replicate in human cells
Didier Trono and Luigi Naldini
Salk Institute

The FDA and NIH created new programs—the Gene Therapy Clinical Trial Monitoring
Plan and the Gene Transfer Safety Symposia—in an effort to ensure the safety and
transparency of gene therapy clinical trials. This was after the death of an 18-year-old
1999 patient during a clinical trial using an adenovirus vector. Additional patient protection
caused delays in research at the time, but has led to greater emphasis on safety and
data sharing in gene therapy research efforts since
University of Pennsylvania, Food and Drug
Administration (FDA), National Institutes of
Health (NIH)
A clinical trial of gene therapy using a gamma retrovirus raised concern about the safety of
gene insertion
Ten patients with X-linked severe combined immunodeficiency (SCID) were
2000 treated with gene therapy. While 9 out of 10 were treated, 4 of the 9 patients
developed leukemia. This study demonstrated the need for improved viral vectors in
gene therapy Alain Fisher and Marina Cavazzana-Calvo
Necker Hospital for Sick Children
 The FDA approved the first clinical trial (in humans) using an LVV to test the
2002 safety and tolerability of a single infusion in patients with HIV. The phase 1 trial
was successfully completed, opening the door for more LVV research including
a phase 2 trial Rob Roy MacGregor
University of Pennsylvania

The National Medical Products Administration, formerly the China Food and
2003 Drug Administration, approved the world’s first commercially available gene
therapy to treat squamous cell carcinoma, a form of skin cancer
National Medical Products Administration
China

In a clinical trial, a genetic eye disease was treated using an adeno-associated

2009 virus (AAV) vector. Eight years later, this pivotal trial led to the FDA approval of
the first gene therapy in the United States Jean Bennett
University of Pennsylvania

The first engineered TAL-effector nucleases were described with the

2010 ability to cause targeted mutagenesis Michelle Christian, Tomas Cermak, and Erin L. Doyle
University of Minnesota and Iowa State University
 A self-inactivating LVV was first used in clinical trials of gene addition
therapy in hemoglobinopathies
2010
Philippe Leboulch
Paris Descartes University
The European Medicines Agency (EMA) approved the first AAV-based gene
addition therapy for the treatment of lipoprotein lipase deficiency (LPLD)
2012 This gene therapy was later removed from the market in 2017 due to its
limited use European Medicines Agency (EMA)
Europe

Scientists developed a gene-editing technique called CRISPR/Cas9 that

2012 can modify specific DNA sequences
Jennifer Doudna, Emmanuelle Charpentier, and team
UC Berkeley

The EMA approved the first gamma retrovirus-based gene addition therapy to treat
2016 adenosine deaminase severe combined immunodeficiency (ADA-SCID).
European Medicines Agency (EMA)
Europe
 The FDA approved the first in vivo gene addition therapy to treat
2017 patients with a rare form of inherited blindness called biallelic
RPE65 mutation-associated retinal dystrophy US Food and Drug Administration (FDA)
United States
The FDA approved a CAR T-cell therapy for the treatment of patients
with relapsed/refractory large B cell lymphoma (R/R DLBCL)
2017 Approved by the EMA in 2019 US Food and Drug Administration (FDA)
United States

The first clinical trial using CRISPR/Cas9 was initiated. This study is
2018 investigating the use of CRISPR/Cas9 for gene disruption in beta
Stanford University, Columbia University, The Children’s Hospital at TriStar
hemoglobinopothies Centennial Medical Center, and more

The FDA approved an AAV-based in vivo gene addition therapy for spinal muscular
2019 atrophy
Conditionally approved by the EMA in 2020
US Food and Drug Administration (FDA)
United States
 The EMA approved an LVV-based ex vivo gene addition therapy for
2019 a genetic disease called transfusion-dependent beta-thalassemia
(TDT)
European Medicines Agency (EMA)
Europe

FDA finalizes 6 gene therapy guidelines including draft guidelines for the
2020 research and clinical development of gene therapies
US Food and Drug Administration (FDA)
United States
4-day-old newborn received in vivo gene therapy for spinal muscular
atrophy, making her the youngest patient at this point to receive gene
2021
therapy
Charlotte Hollman
Woman’s Hospital, Baton Rouge, LA, USA
types

Gene addition Gene editing

Gene addition treats Gene editing treats

diseases at the genetic diseases at the genetic
level by adding genetic level by directly modifying
material to a person’s a patient’s DNA through a
cells to compensate for number of different
a missing or faulty techniques.
gene.
 types

How does geneGene

addition
addition
work?
• Gene addition is a common gene therapy technique being explored for
single-gene diseases—disorders where a mutation occurs in one or both sets
of your genes.

• This gene therapy technique usually involves the insertion of functional (or
healthy) copies of a gene (otherwise known as a transgene) into a person’s
cells by way of a vector
 gene addition
• Vectors deliver the functional gene
to the patient’s cells, either in
vivo or ex vivo. Once inside the
cell, the transgene provides the cell
with instructions that lead to the
production of functional proteins.

• With gene addition therapy, the

mutated gene does not need to be
replaced or removed.

• This provides the cell with the

instructions that lead to the
production of functional genes,
 types

How does geneGene

addition
editing
work?
• Gene editing involves the creation of targeted breaks in the DNA,
with or without instructions to repair them, through a number of
different techniques.

• There are 2 primary techniques in gene editing:

disruption/inactivation, correction/insertion.
 gene editing
“Disrupting” or “inactivating”
genetic material that is responsible
for the genetic disease. This can be
achieved by turning off genes that
cause disease or disrupting a
separate gene that will compensate
for the disease-causing gene

“Correcting” genetic material by

creating a break in the gene and
providing a corrective template or
“inserting” new genetic material
for the cell to use to repair the
mutated gene
 METHODS
How genetic material is delivered to cells?
In vivo Ex vivo
● Genetic material can ● Genetic material can be
be delivered to target delivered to a person’s
cells that remain inside cells after they are taken
a person’s body (this is out of their body (this is
called in vivo) called ex vivo)
 methods
In vivo delivery
Refers to direct delivery of genetic material
either intravenously (through an IV) or
locally to a specific organ (eg, directly into
the eye).

Works through the help of a vector, which directly

inserts functional copies of a gene into target cells
to treat a mutated or missing gene
 methods
Ex vivo delivery
Refers to the process of removing specific
cells from a person, genetically altering them
in a laboratory, and then transplanting them
back into the person.

Works by genetically modifying a patient’s

stem cells, which then replace target cells that
have a missing or malfunctioning gene

Today, ex vivo gene therapy is most frequently

applied to hematopoietic stem cells (HSCs);
this is used to treat blood and immunological
diseases as well as genetic diseases that
affect tissues and organs that can be reached
by blood cells
 Vectors
What is a vector?

● Gene therapy works by introducing genetic material (such as

a transgene or nuclease) into the nucleus of the cell.

● The system used to deliver genetic material is known as a vector. Think

of a vector as a microscopic delivery truck that transports packages
(genetic material) to specific locations (target cells).

Types of vectors
● Viral and Non-viral
Vectors
CLINICAL TRIAL

PRECLINICAL PHASE 1 PHASE 2 PHASE 3

Lab Studies Human Safety Expanded Safety Efficacy & Safety

 Clinical trial

Pre clinical
Before a gene therapy is tested in clinical trials, researchers
must study it first in a laboratory. These studies are called
preclinical studies, and help researchers decide what gene
therapies are ready to put into clinical trials. When a gene
therapy is ready for a clinical trial in people living with a
specific disease, the manufacturer submits an application to
the FDA to gain approval to start this type of clinical trial.4
There are usually 3 phases to each clinical trial, each
involving a slightly greater number of people.
Clinical trial

Phase 1 Phase 2 Phase 3

The clinical study is
The clinical study The clinical study conducted in a larger
is conducted in a is conducted in a group of people with a
small group of group of people disease or condition.
healthy people to with a specific Most phase 3 studies are
test the drug for disease or used to compare the
safety and condition to test the efficacy and safety of the
determine its drug’s safety and new treatment to the
appropriate dose, efficacy (how well current standard of care.
These studies tend to last
dosing schedule, it works).
the longest and usually
and method of . become the basis for
Clinical trial

How long does it take to get a gene therapy approved?

It can take many years (even decades) for a gene therapy to move
from discovery, through preclinical and clinical trials, and into
approval (where it is available for public use).
Variables that affect the time it takes to get a gene therapy approved
include:
• study planning,
• patient enrollment,
• time to approve trial,
• ethics reviews, and funding.
applications
Approved Gene Therapies
● After a long history of research, gene therapy is now being used in clinical
practice
risks
Potential risks of gene therapy include:
risks
Potential risks of gene therapy include:
 Ethical issues
Because gene therapy involves making changes to the body’s
set of basic instructions, it raises many unique ethical
concerns. The ethical questions surrounding gene therapy
include:
1. How can “good” and “bad” uses of gene therapy be
distinguished?
2. Who decides which traits are normal and which constitute a
disability or disorder?
3. Will the high costs of gene therapy make it available only to
the wealthy?
4. Could the widespread use of gene therapy make society less
 Ethical issues

Current gene therapy research has focused on treating

individuals by targeting the therapy to body cells such as
bone marrow or blood cells. This type of gene therapy
cannot be passed to a person’s children. Gene therapy
could be targeted to egg and sperm cells (germ cells),
however, which would allow the inserted gene to be
passed to future generations. This approach is known as
germline gene therapy.
 Ethical issues

The idea of germline gene therapy is controversial. While it

could spare future generations in a family from having a
particular genetic disorder, it might affect the development
of a fetus in unexpected ways or have long-term side
effects that are not yet known. Because people who would
be affected by germline gene therapy are not yet born,
they can’t choose whether to have the treatment. Because
of these ethical concerns, the U.S. Government does not
allow federal funds to be used for research on germline
gene therapy in people.
cloning
 introduction

Cloning is a technique scientists use to make exact genetic copies of

living things. Genes, cells, tissues, and even whole animals can all be
cloned.
The cloning of animals has been used in a number of different
applications. Animals have been cloned to have gene mutations that
help scientists study diseases that develop in the animals. Livestock
like cows and pigs have been cloned to produce more milk or meat.
Clones can even “resurrect” a beloved pet that has died. In 2001, a cat
named CC was the first pet to be created through cloning. Cloning
might one day bring back extinct species like the woolly mammoth or
giant panda.
 BACKGROUND
Lost in the midst of all the buzz about cloning is the fact that cloning is nothing new: its rich scientific
history spans more than 100 years. The landmark examples below will take you on a journey through
time, where you can learn more about the history of cloning.
1885 First-ever demonstration of artificial embryo twinning

1902 Artificial embryo twinning in a vertebrate

1928 The cell nucleus controls embryonic development

1952 First successful nuclear transfer

BACKGROUND

1958 Nuclear transfer from a differentiated cell

1975 First mammalian embryo created by nuclear transfer

1984 First mammal created by nuclear transfer

1987 Nuclear transfer from embryonic cell

BACKGROUND

1996 Nuclear transfer from laboratory cells

1996 Dolly: First mammal created by somatic cell nuclear transfer

1997 First primate created by embryonic cell nuclear transfer

1997 Nuclear transfer from genetically engineered

laboratory cells
BACKGROUND
1998- More mammals cloned by somatic cell nuclear transfer
1999

2001 Endangered animals cloned by somatic cell nuclear transfer

Primate embryonic stem cells created by somatic cell nuclear

2007 transfer

2013 Human embryonic stem cells created by somatic

cell nuclear transfer
 types

Therapeutic
Gene cloning Reproductive cloning cloning

Creates copies of genes Creates copies of whole Creates embryonic stem

or segments of DNA
animals. cells. Researchers hope to
use these cells to grow
healthy tissue to replace
injured or diseased tissues
in the human body.
 types

How does gene cloning

Gene CLONING
work?
• The basic steps in gene cloning are:
1. DNA extracted from an organism known to have the gene of interest is cut into
gene-size pieces with restriction enzymes.
2. Bacterial plasmids are cut with the same restriction enzyme.
3. The gene-sized DNA and cut plasmids are combined into one test tube. Often, a
plasmid and gene-size piece of DNA will anneal together forming a recombinant
plasmid(recombinant DNA).
4. The recombinant plasmids are transferred into bacteria using electroporation or
heat shock.
types

Gene CLONING
5. The bacteria is plated out and allowed to grow into colonies. All the colonies on all
the plates are called a gene library.

6. The gene library is screened to identify the colony containing the gene of interest
by looking for one of three things:
• the DNA sequence of the gene of interest or a very similar gene
• the protein encoded by the gene of interest
• a DNA marker whose location has been mapped close to the gene of
interest
 types

Reproductive CLONING
Reproductive cloning is defined as the deliberate production of
genetically identical individuals. Each newly produced individual is a
clone of the original. Monozygotic (identical) twins are natural
clones. Clones contain identical sets of genetic material in the
nucleus—the compartment that contains the chromosomes—of
every cell in their bodies. Thus, cells from two clones have the
same DNA and the same genes in their nuclei.
 types

Reproductive CLONING
All cells, including eggs, also contain some DNA in the energy-
generating “factories” called mitochondria. These structures are in
the cytoplasm, the region of a cell outside the nucleus. 
Mitochondria contain their own DNA and reproduce
independently. True clones have identical DNA in both the nuclei
and mitochondria, although the term clones is also used to refer to
individuals that have identical nuclear DNA but different
mitochondrial DNA
 types

therapeutic CLONING
Therapeutic cloning is the transfer of nuclear material isolated from a
somatic cell into an enucleated oocyte in the goal of deriving
embryonic cell lines with the same genome as the nuclear donor.
Somatic cell nuclear transfer (SCNT) products have histological
compatibility with the nuclear donor, which circumvents, in clinical
applications, the use of immunosuppressive drugs with heavy side-
effects.
 types

therapeutic CLONING
The purpose of therapeutic cloning is to generate and direct the
differentiation of patient-specific cell lines isolated from an embryo
not intended for transfer in utero. Therapeutic cloning, through the
production of these autologous nuclear-transfer embryonic stem
cells (ntESC), offers great promises for regenerative and
reproductive medicine, and in gene therapy, as a vector for gene-
delivery.
 METHODS
How Is Cloning Done?
Artificial Embryo Somatic Cell
Twinning
Nuclear
Transfer
 methods

Artificial Embryo Twinning

Artificial embryo twinning is a relatively low-tech way to make clones.
As the name suggests, this technique mimics the natural process
that creates identical twins.
In nature, twins form very early in development when the embryo
splits in two. Twinning happens in the first days after egg and
sperm join, while the embryo is made of just a small number of
unspecialized cells. Each half of the embryo continues dividing on
its own, ultimately developing into separate, complete individuals.
Since they developed from the same fertilized egg, the resulting
individuals are genetically identical..
 methods

Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT), also called nuclear transfer, uses
a different approach than artificial embryo twinning, but it
produces the same result: an exact genetic copy, or clone, of an
individual. This was the method used to create Dolly the Sheep.
 methods

What does SCNT mean? Let's take it apart:

Somatic cell:
A somatic cell is any cell in the body other than sperm and
egg, the two types of reproductive cells. Reproductive
cells are also called germ cells. In mammals, every
somatic cell has two complete sets of chromosomes,
whereas the germ cells have only one complete set.
Nuclear:
The nucleus is a compartment that holds the cell's DNA.
The DNA is divided into packages called chromosomes,
 methods

Nuclear:
The nucleus is a compartment that holds the cell's DNA.
The DNA is divided into packages called chromosomes,
and it contains all the information needed to form an
organism. It's small differences in our DNA that make
each of us unique.
 methods

Transfer:
Moving an object from one place to another. To make Dolly,
researchers isolated asomatic cell from an adult female sheep. Next
they removed the nucleus and all of its DNA from an egg cell. Then
theytransferred thenucleus from the somatic cell to the egg cell.
After a couple of chemical tweaks, the egg cell, with its new
nucleus, was behaving just like a freshly fertilized egg. It developed
into an embryo, which was implanted into a surrogate mother and
carried to term. (The transfer step is most often done using an
electrical current to fuse the membranes of the egg and the
somatic cell.
 Pros of cloning

1. It can help prevent the extinction of species.

As many organisms in the planet approach endangerment

and extinction, cloning appears to be a possible solution to
restore populations. By utilizing the genetic material of
already dead organisms, cloning can even contribute to
expanding the diversity of gene pools.
 Pros of cloning

2. It can help increase food production

Another major advantage of cloning is that it can serve as

a means to increase agricultural production, particularly
livestock and fresh produce. By manipulating their
biological processes, existing traits of interest are ensured
with the absence of the genetic “lottery” and random
arrangements in the genes during meiosis.
 Pros of cloning

3. It can help couples who want to have children

Last but not the least is the use of cloning as a means to

produce children for infertile and same-sex couples.
Normally, couples would want to have children that are
biologically theirs. Interestingly, the genetic manipulations
to be done could now be targeted at giving the children
the genetic traits of both of their parents.
 Cons of cloning

1. The process is not entirely safe and accurate

Despite being genetically identical with each other, clones

will not be the same regarding behavioral attributes. Aside
from that, their similarities regarding physical appearance
are not guaranteed.
 Cons of cloning

2. The offspring lack genetic uniqueness

Majority of the concerns against cloning are based on the

absence of unique nuclear DNA in the child’s genome.
However, as mentioned above, the notion that cloning
would only result in the absence of genetic uniqueness
would no longer be justifiable, assuming that the
procedures are conducted safely.
 Cons of cloning

3. It is not yet fully-developed

To date, many scientists will agree that the process of

cloning is not yet fully developed to be used as a way to
promote the conservation of species. In fact, some
researchers do not even acknowledge cloning as it fails to
recognize the main drivers of extinction in the first place:
the destruction of habitats and hunting.
 Cons of cloning

4. It is regarded as unethical, and the probability of abuse

is very high

One of the strongest arguments against cloning is

about its ethical concerns. Aside from the idea of
manipulating living organisms, critics say that the mere
process of clone production requires the exploitation
of life.
 Ethical issues

• Gene cloning is a carefully regulated technique that is

largely accepted today and used routinely in many labs
worldwide. However, both reproductive and therapeutic
cloning raise important ethical issues, especially as
related to the potential use of these techniques in
humans.
 Ethical issues
• Reproductive cloning would present the potential of
creating a human that is genetically identical to another
person who has previously existed or who still exists. This
may conflict with long-standing religious and societal
values about human dignity, possibly infringing upon
principles of individual freedom, identity and autonomy.
However, some argue that reproductive cloning could help
sterile couples fulfill their dream of parenthood. Others see
human cloning as a way to avoid passing on a deleterious
gene that runs in the family without having to undergo
embryo screening or embryo selection.
 Ethical issues

• Therapeutic cloning, while offering the potential for

treating humans suffering from disease or injury, would
require the destruction of human embryos in the test
tube. Consequently, opponents argue that using this
technique to collect embryonic stem cells is wrong,
regardless of whether such cells are used to benefit sick
or injured people.
 https://www.bioexplorer.net/pros-and-cons-of-cloning.html/

https://www.bioexplorer.net/bioethical-issues.html/#4_Cloning

https://learn.genetics.utah.edu/content/cloning/whatiscloning/

https://www.genome.gov/about-genomics/fact-sheets/Cloning-Fact-Sheet

https://www.thegenehome.com/gene-therapy-process/examples
Thank you!