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In conventional treatments of gene therapy viral and nonviral vectors are commonly used for the delivery of the
gene.
 These are used to deliver normal copies of a gene into a
cell that tends to contain mutated copies of a gene.
 However there are times that when you do add the good
copy of the gene it might not work.

Dominant negative:

For example there are certain cases when a mutated gene
might produce a protein that prevents the normal protein from
doing its job and in this case if you simply add the normal
gene it won’t help. Mutated genes that work this way are
called dominant negative.
How do we then deal with a dominant negative?
 In this situation one could either repair the product of the
mutated gene or they could get rid of it altogether.
 Some new methods have been developed by scientists
which serve as potential approaches to gene therapy.
 Every technique being used for this purpose requires an
efficient and specific means of delivering the gene to the
target cells.
 Some of these are
1.
SMaRT
2. Triple-helix forming oligonucleotides
3. Antisense
4. Ribozymes
A technique for repairing mutations: SMaRT:

SMaRT stands for spliceosome-mediated RNA
Trans-splicing.

This technique tends to target and repair the
messenger RNA transcripts that have been
copied from the mutated gene.

Instead of replacing the entire gene this
technique tends to repair a particular section of
the mRNA that contains the mutation.
SMaRT involves three steps
1) Delivery of a RNA strand that pairs specifically
with the intron next to the mutate segment of
mRNA. Once bound, this RNA strand prevents
spliceosomes from including the mutated segment
in the final, spliced RNA product.
2) Simultaneous delivery of a correct version of the
segment to replace the mutated piece in the final
mRNA product
3) Translation of the repaired mRNA to produce the
normal, functional protein
Techniques to prevent production of a mutated protein:
Triple-helix forming oligonucleotides
 Triple-helix-forming oligonucleotide gene therapy
targets the DNA sequence of a mutated gene to
prevent its transcription.
 This technique involves the delivery of short, singlestranded pieces of DNA, called oligonucleotides, that
bind specifically in the groove between the double
strands of the mutated gene's DNA.
 Binding produces a triple-helix structure that
prevents that segment of DNA from being transcribed
into mRNA.
Antisense gene therapy aims to turn off a
mutated gene in a cell by targeting the mRNA
transcripts copied from the gene.
 Antisense gene therapy involves the following
steps:
 Delivery of an RNA strand containing the
antisense code of a mutated gene
 Binding of the antisense RNA strands to the
mutated sense mRNA strands, preventing the
mRNA from being translated into a mutated
protein

Like antisense, ribozyme gene therapy aims to turn
off a mutated gene in a cell by targeting the mRNA
transcripts copied from the gene. This approach
prevents the production of the mutated protein.
 Ribozyme gene therapy involves the following
steps:
 Delivery of RNA strands engineered to
function as ribozymes.
 Specific binding of the ribozyme RNA to
mRNA encoded by the mutated gene
 Cleavage of the target mRNA, preventing it
from being translated into a protein
Protein therapy





Therapeutic
Gene therapy
proteins
are
used
to 
medically treat a disease.
form of protein therapy.
They are used for a wide array of 
Instead of the therapeutic usage of the protein
diseases
itself, genes are used.
In these cases the protein is either 
Gene therapy works by placing into a cell a
lacking or deficient, or the therapeutic
defined gene to either replace a defective gene
protein is used to inhibit a biological
or to increase the amount of a specific gene in
process.
a targeted cell/tissue
Protein
therapy
uses
well
defined, 
This is done in order to produce a higher
precisely structured proteins
amount of the desired protein.
The optimal doses of individual protein 
To deliver the therapeutic gene either a carrier
for a particular treatment are already
(vector DNA) must be used
defined

Gene therapy can actually be considered a

Or the therapeutic DNA must be introduced as
Also the biological effects are well known
“naked” DNA, most often as plasmid DNA, into
in this case.
the target cells.
There are still serious, unsolved problems related to gene
therapy including:
1. Difficulty integrating the therapeutic DNA (gene) into
the genome of target cells
2. Risk of an undesired immune response
3 Potential toxicity, immunogenicity, inflammatory
responses and oncogenesis related to the viral vectors; and
4. The most commonly occurring disorders in humans such
as heart disease, high blood pressure, diabetes, Alzheimer’s
disease are most likely caused by the combined effects of
variations in many genes, and thus injecting a single gene
will not be beneficial in these diseases.
The benefits of protein therapy include:

Using a human protein with no immunogenic response

No need for viral vectors

Localized effect at the target tissue, and

Predictability of dose.

On the other hand, an obstacle of protein therapy is the
mode of delivery: oral, intravenous, intra-arterial, or
intramuscular routes of the protein’s administration are
not always as effective as desired; the therapeutic protein
can be metabolized or cleared before it can enter the
target tissue.

It seems that protein therapy will become the treatment
modality of choice for many disorders for at least the
next 10 years—at least until further research has resolved
the hurdles and risks related to gene therapy.

Many unique technical and ethical considerations have
been raised by this new form of treatment

Several levels of regulatory committees have been
established to review each gene therapy clinical
trial prior to its initiation in human subjects.

Ethical considerations include
a)
deciding which cells should be used
b)
how gene therapy can be safely tested and evaluated in
humans
c)
what components are necessary for informed consent
d)
and which diseases and/or traits are eligible for gene
therapy research.
Germ line gene therapy is difficult as stable integration
and gene expression requires gene replacement or repair;
however currently only gene addition can be done.
 Gene addition could result in insertional mutations and
productions of chimeras
 Genetic enhancement is another issues which could be
misused by totalitarian governments
 Also as it tends to be expensive only a certain class can
avail the treatment.
 The treatment can cause unintended consequences and
might affect evolution to a greater degree.
 Germ line modifications tend to pose a risk to future
generations.

This study was conducted on 6 patients in California
 A person with HIV who didn't take antiretroviral drugs
for three months remained free of the virus, thanks to
a groundbreaking gene therapy.
 The success raises the prospect of keeping HIV in check
permanently without antiretrovirals.
 The gene therapy works by locking the virus out of the
CD4 white blood cells it normally infects.
 In this small phase I study they had one virus-free patient
and 10-fold reductions in another two.

Zinc fingers:
 To deliver the treatment, doctors remove blood from the
patient and isolate CD4 and other white blood cells.
 Specialised molecular "scissors" called zinc finger
proteins enter the cells and sabotage a gene called CCR5,
which makes a protein that helps HIV to enter cells.
 It is unclear what role CCR5plays normally, although
researchers know that cells can survive without it – and
will remain uninfected by HIV.
 These cells are then returned to the patient in the hope
that they will multiply and provide a permanent source of
cells immune to HIV, potentially locking out HIV
completely.
Double sabotage
 The secret to making the treatment work best, according
to research, is therefore to eliminate both genes that
make CCR5 in as many cells as possible. If only one is
sabotaged, cells can still make enough CCR5 protein to
allow the virus to invade. In doubly sabotaged or "biallelic" cells, there is no way in.