GENE THERAPY Medical biotechnology - muhammad1988adeel

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23 Οκτ 2013 (πριν από 3 χρόνια και 10 μήνες)

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In conventional treatments of gene therapy
viral and non
-
viral 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, single
-
stranded 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

Gene

therapy


Therapeutic

proteins

are

used

to

medically

treat

a

disease
.


They

are

used

for

a

wide

array

of

diseases



In

these

cases

the

protein

is

either

lacking

or

deficient,

or

the

therapeutic

protein

is

used

to

inhibit

a

biological

process
.


Protein

t桥rapy

畳us

well

defi湥d,

precisely

structured

proteins



T桥

optimal

do獥s



i湤ividual

protein

for

a

partic畬ar

treatme湴

are

already

defined



Al獯

t桥

biological

effects

are

well

known

in

this

case
.




Gene

therapy

can

actually

be

considered

a

form

of

pro
tein

therapy
.


Instead

of

the

therapeutic

usage

of

the

protein

itself,

genes

are

used
.


Gene

therapy

works

by

placing

into

a

cell

a

defined

gene

to

either

replace

a

defective

gene

or

to

increase

the

amount

of

a

specific

gene

in

a

targeted

cell/tissue



Th楳



d潮e



潲o敲



pr潤uce

a

h楧h敲

amount

of

the

desired

protein
.




d敬ev敲

the

th敲ap敵tic

g敮e

敩eh敲

a

carrier

(v散t潲

D乁)

must



us敤



Or

the

therapeutic

DNA

must

be

introduced

as

“naked”

DNA,

most

often

as

plasmid

DNA,

into

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, immu
nogenicity, 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 immuno
genic 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,

intrave
nous,

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


S
everal
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

CCR5
plays 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 "bi
-
allelic" cells, there is no way in.