notes about the recently emerging field of gene therapeutics, it includes the steps , processes , components and types of gene therapy

1. GENE THERAPY
Gene therapy, a cutting-edge medical technique, has emerged as a game-changer
in the field of healthcare. This transformative approach involves the modification
of a patient's genetic material to treat or prevent a range of diseases. Let's
embark on an in-depth exploration of the technical intricacies of this
revolutionary treatment, its historical evolution, and its profound implications
for the future of medicine.
It is introduction of a normal gene into an individual’s genome in order to repair
a mutation that causes a genetic disease.
Gene therapies are designed to be one-time treatments that target the genetic
root cause of diseases.
HISTORY
The concept of gene therapy arose during the 1960s and 1970s and it is still in its
infancy, meaning that there is a paucity of reliable, long-term data on the safety
and efficacy of this therapy. The first attempt at modifying human DNA was
performed in 1980 by Martin Cline, but the first successful nuclear gene transfer
in humans, approved by the National Institutes of Health, was performed in May
1989.[3] The first therapeutic use of gene transfer as well as the first direct
insertion of human DNA into the nuclear genome was performed by French
Anderson in a trial starting in September 1990.
TYPES OF GENE ALTERATIONS

2. 1) Gene addition
It involves the introduction of a new gene into the body to target a specific aspect
of what causes the disease. Gene addition delivers a new gene into the body to
target a specific aspect of what causes disease and can supplement another
medication that targets that same aspect to help it work better at treating that
disease.
2) Gene inhibition
Gene inhibition involves deactivating or “silencing” the expression of a mutated
or faulty gene that codes for a toxic protein or too much protein.
3) Gene editing
The mutant gene that is causing disease is edited in order to correct the mutation.
This technique aims to repair the altered gene by inserting, removing, or
changing specific pieces of a person's existing DNA.
4) Gene replacement
Gene replacement is a way to treat genetic diseases. It replaces the function of a
missing, faulty, or nonworking gene with a new, working copy of the
malfunctioning gene. The new gene sits inside the nucleus, or control center, of
cells and allows the cells to produce the missing proteins that are critical for the
body to function. Viruses are used in gene replacement because of their natural
ability to enter into the cells of the body. In gene replacement, scientists alter or
reengineer a virus so it can be used as a vector, or delivery vehicle, without
causing disease in humans.
The difference between gene addition and gene replacement:
What may be confusing about gene replacement and gene addition (because they
sound similar) is that, in gene replacement, a gene that is normally found in the
body is added and with gene addition, a gene that is novel to the body is added.
KEY COMPONENTS OF GENE THERAPY
Genes
A new, working copy of the human gene is deliver to the cell, allowing the body to

3. make the protein that is missing or in short supply. This new gene is create in a
laboratory and is specific to the disease being treated. That is, scientists work to
discover which gene needs to be replaced and figure out how to create the new,
working gene.
Vectors
Vectors are the delivery vehicles used to carry a new, working copy of the
missing or nonworking gene into the right cells inside the body. Viruses are used
because they are very good at getting inside of cells and carry the new working
genes into the nucleus of the cell.
There are two systems for the delivery of transgene into the cell – viral and
non-viral.
The non-viral approaches are represented by polymer nanoparticles, lipids,
calcium phosphate, electroporation/nucleofection or biolistic delivery of
DNA-coated microparticles. The safety is mentioned as the major advantage of
non-viral approaches. In general, non-viral delivery of transgene is less effective
in comparison to viral systems.
There are several categories of viral vectors. We distinguish two main types of
vectors depending on whether the DNA is integrated into chromatin of the host
cell or not. Retroviral vectors derived from gammaretroviruses or lentiviruses
persist in the nucleus as integrated provirus and reproduce with cell division.
Other types of vectors (e.g. those derived from herpesviruses or adenoviruses)
remain in cell in the episomal form.
The overview of viral vectors is depicted in Figure 2(above). Adenoviral and
retro/lentiviral vectors are most frequently used in research and gene therapy

4. clinical trials.
Let us go in further details of these vectors-
1) adenoviral vectors are very popular.
They have been used for several decades. Since adenoviruses are non-enveloped
dsDNA viruses, they are relatively resistant to chemical and physical agents,
which enable them to persist out of host cells and make the work in laboratory
easier in comparison to enveloped RNA viruses. They are often used in cancer
gene therapy as replication-defective or replication-competent vectors. They
infect proliferating as well as non-dividing cells. In general, adenoviral vectors
are considered safe. Since they do not integrate into host DNA, the transduction
is transient.
The drawback is their immunogenicity(the ability of cells/tissues to provoke an
immune response)
2) Adeno-associated vectors share with retroviral vectors the ability to integrate
into host DNA. Wild type adeno-associated virus integrates into specific site of
the chromosome 19. Recombinant vectors lack this characteristic and the risk of
insertional mutagenesis exists. These vectors transduce dividing and
non-dividing cells, and the transgene expression is long term. Transduced cells
are minimally immunogenic
3) Herpetic viruses are relatively complex enveloped dsDNA viruses. The vectors
have been prepared from Herpes simplex type 1 virus.
They are less immunogenic in comparison to adenoviruses. The transduction is
transient.
the drawback of HSV 1-derived vector is the short-term expression of the
transgene. Herpes virus-derived vectors are preferentially used in vaccination
Vectors derived from alfaviruses (ssRNA viruses) are also used in cancer gene
therapy and immunotherapy
4. Retroviral vectors
Retroviruses are relatively complex enveloped RNA viruses with diploid ssRNA
genome. Typical feature of retroviruses and retroviral vectors is their ability to
integrate into host DNA. Viral RNA is reversibly transcribed and integrated in
the form of provirus. They very effectively cooperate with enzymes of the host
cell, and they use it for their own replication and long-term expression of viral
proteins. The entry of virus into the host cell is receptor-dependent
Many types of retroviruses (bovine leukaemia virus, Rous sarcoma virus,
lentiviruses and spumaviruses) were used for preparation of vectors. The most
popular vectors are constructs based on MoMLV and HIV.
PHYSICAL METHODS OF GENE INSERTION

5. Direct injection:
Injection directly into skeletal of cardiac muscle can be effective in expression of
some recombinant genes.
formation of myotubes and transfer of DNA
- no evidence of DNA integration but expression persisted for 6 months
Microparticle bombardment (gene gun):
DNA adsorbed onto submicron sized gold or tungsten particles. Particles
accelerated to high velocity using a gas discharge
- Expression observed following bombardment of skin, muscles, liver, intestine
and mammary gland
- may be particularly useful for gene vaccination
similar technique can be used on cells
Electroporation:
Cells exposed to very high electric field strengths in the presence of gene or
plasmid
Blasts temporary holes in cell membrane
Allows passage of DNA into the cytoplasm
STEPS IN GENE THERAPY
1) Creating a working gene
The gene transfer therapy involves creating a working (or functional) gene in the
laboratory. The working gene contains the instructions for making a needed
protein.
2) Building a therapeutic vector
The working gene now has to be delivered into the body. The shell of the virus is
created without the viral DNA and working gene is put inside the empty shell. No
longer is a virus, the therapeutic vector designed to deliver the working gene to
the cells in the body where it is needed.
3) Determining eligibility
As part of gene therapy research, a health-care provider must determine whether
a patient is eligible.
4)Delivering the working gene
Once a patient is determined to be eligible, the gene therapy is ready for
administration to evaluate its safety and impact
5)Monitoring safety and efficacy
Regular monitoring after gene therapy is important because it allows researchers
to understand any risk and what impact the gene transfer is having. As with all

6. medications, responses to gene therapy may vary.
Researchers are testing several approaches to gene therapy, including:
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.
GERMLINE THERAPYAND SOMATIC CELL GENE THERAPY
Germline therapy
Germline therapy involves the
modification of the genes inside germ
or gamete cells, which include sperm or
ova. Once fused together, the zygote
divides and passes on the modified
gene to all other cells of the body
during the development of offspring. In
this way, germline therapy alters the
genome of future generations to come.
Somatic gene therapy
Unlike germline therapy, somatic gene
therapy involves the insertion of
therapeutic DNA into body cells, rather
than germ cells or gametes. The field of
somatic gene therapy is surrounded by
fewer ethical issues as compared to
germline gene therapy. While this may
be true, this therapeutic approach
remains in the early stages of
development.

7. IN VIVO AND EX-VIVO APPROACHES
CHALLENGES FACED BY GENE THERAPY
1) Delivering the gene to the right place and switching it on:
it is crucial that the new gene reaches the right cell
delivering a gene into the wrong cell would be inefficient and could also cause
health problems for the patient
even once the right cell has been targeted the gene has to be turned on
cells sometimes obstruct this process by shutting down genes that are showing
unusual activity.
2)Avoiding the immune response:
The role of the immune system is to fight off intruders.
Sometimes new genes introduced by gene therapy are considered
potentially-harmful intruders.
This can spark an immune response in the patient, that could be harmful to
them.

8. Scientists therefore have the challenge of finding a way to deliver genes without
the immune system ‘noticing’.
This is usually by using vectors that are less likely to trigger an immune
response.
3) Making sure the new gene doesn’t disrupt the function of other genes:
Ideally, a new gene introduced by gene therapy will integrate itself into the
genome of the patient and continue working for the rest of their lives.
There is a risk that the new gene will insert itself into the path of another gene,
disrupting its activity.
This could have damaging effects, for example, if it interferes with an important
gene involved in regulating cell division, it could result in cancer.
4) The cost of gene therapy:
Many genetic disorders that can be targeted with gene therapy are extremely
rare.
Gene therapy therefore often requires an individual, case-by-case approach. This
may be effective, but may also be very expensive.
Ethical and safety concerns
Some aspects of gene therapy, including genetic manipulation and selection,
research on embryonic tissue, and experimentation on human subjects, have
aroused ethical controversy and safety concerns.
On the other hand, others have argued that genetic engineering may be justified
where it is consistent with the purposes of God as creator. Some critics are
particularly concerned about the safety of germline gene therapy, because any
harm caused by such treatment could be passed to successive generations.
Benefits, however, would also be passed on indefinitely. There also has been
concern that the use of somatic gene therapy may affect germ cells.