DNA Based Diagnosis and Treatment

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14 Δεκ 2012 (πριν από 4 χρόνια και 8 μήνες)

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DNA Based Diagnosis

and Treatment

Elsa
Chien
, Wayne (Kin) Chun,
Necole

Chung and Silky Lee

September 28, 2012

Name:


Sign up Date:


PHM142 Fall 2012


Instructor: Dr. Jeffrey Henderson

What is DNA Based Diagnosis?


detect presence of specific nucleic acid
associated with a disease


high sensitivity and specificity, detects target
DNA even at very low concentrations


tests don’t show pathogen viability


may not detect if pathogens and viruses mutate
and change genetic structure


What is DNA Based Diagnosis?
Cont’d . . .


false negatives may be caused
by inaccurate selection of tissue
sources


diagnose conditions such as
cancer, cystic fibrosis and Down
syndrome and infectious
diseases such as anthrax and
chlamydia


Source:
http://www.cartoonstock.com/newscartoons/cartoonists/aba/lowres/
aban55l.jpg

Polymerase Chain Reaction


series of repetitive replication
cycles


uses
thermostable

DNA
polymerase to make several
million copies of a targeted
sequence


detects short deletions or
insertions


Target amplification


DNA/RNA duplicated


Source: http://www2.le.ac.uk/departments/emfpu/genetics/explained/pcr

Southern Blot Hybridization


DNA fragments separated by
electrophoresis, then blotted
on nylon filter


blotted nucleic acids fixed and
hybridized using radioactively
labelled
ssDNA


used to diagnose genetic
diseases from mutations


inheritance of a disease can
also be predicted by
polymorphic markers


Source: http://www.molecularstation.com/dna/southern
-
blot/


Uses mRNA as starting
material


detects rearrangements
within the coding region


Can be used to detect
either large gene
alterations or point
mutations and
polymorphisms altering
a restriction site


Source: http://
www.molecularstation.com/rna/northern
-
blot
/

Northern Blot Hybridization


Sanger/ Chain Termination


Sequencing


Provides sequence of
nucleotides on target DNA


Compares and identify
microorganisms and
mutations


Primer prepared with DNA
polymerase and fluorescent
dideoxynucleotides

(
ddNTP
)



Source: http://www.bio.davidson.edu/courses/bio111/seq.html

Shotgun Sequencing

Source: http://www.bio.davidson.edu/COurses/genomics/method/hss2.gif


DNA sequence fragmented,
cloned, sequenced then
aligned


quick and inexpensive


can easily detect
polymorphisms


may take many repeats to
accurately align fragmented
DNA


used to complete the Human
Genome project


Gene Therapy


Use of DNA, to modify gene expression in cells
for a therapeutic effect.


Two types of gene therapy


Somatic gene therapy


Germ line Gene therapy


Purpose of Gene Therapy


Provide a
cure
for various genetic
diseases such as:


Cystic fibrosis


Haemophilia


Thalassemia


Provide an
alternate mode of delivery

of
biological products to the body


Provide new
therapy
for disorders such as:


Cancer


Inflammatory diseases


Artherosclerosis



Germ Line Gene Therapy


gene therapy used to modify the inherited
characteristics of germ cells. Therefore,
descendants will also inherit this
manipulation.


to this date, there has not been any clinical
protocols involving gene transfer to germ
line cells on humans due to ethical debate.


Somatic Gene Therapy


Gene therapy on somatic cells (biological
cells other than reproductive cells i.e.
germ cells)


Can be theoretically:


applied to any disease (as long as molecular
pathogenesis is known)


designed to produce a therapeutic effect for a
finite duration of action via pharmacokinetic
properties


Therefore integration of new genetic material into
the chromosomes does not have to be
permanen


Gene Therapy


Viral Methods


Deliver genetic information to target cells by
ex vivo
or
in vivo


Modified DNA injected into viral vectors


Ex vivo
: target cells extracted from patients,
therapeutic genes inserted and cells returned
to patients


In vivo
: viral vectors introduced to patients,
targeting specific tissues and organs that
require gene transfer

Adenoviruses

Source: http://www.daviddarling.info/encyclopedia/A/adenovirus_infection.html


Non
-
enveloped icosahedral
-
shaped viruses that contain a
linear
dsDNA

genome


Transfer
transgenes

to
various cells, tissues and
organs


Infect dividing and non
-
dividing cells


Large packaging capacity



Life Cycle of Adenoviruses

Source: Warnock,
Daigre

and Al
-
Rubeai
, 2011.


Virus binds to a
coxsackievirus

and adenovirus receptor (CAR) on
host cell surface


Enters cell and releases virus
genome into host nucleus but does
not become integrated


Transcription of viral DNA begins
and transient
transgene

expression occurs

Uses of Adenoviruses



Used for cancer treatment


In normal cells, DNA damage, cell cycle
abnormalities or hypoxia can result in the
development of tumours



Tumour suppressor genes are protective
genes that limit the growth of tumours



Mutated or inactivated tumor suppressor
genes can lead to cancer



In gene therapy, vectors are used to target
cancer cells by transporting the tumour
suppressor gene p53



Overexpression

of p53 inhibits tumour
growth and induces apoptosis


Used to study stem cell differentiation, AIDS,
cardiovascular diseases and pulmonary
tuberculosis


Source: http://en.wikipedia.org/wiki/File:P53_pathways.jpg

Adeno

associated virus (AAV)



Non
-
enveloped,
icosahedral

shaped



Linear
ssDNA

genome


AAV serotype 2 mostly

used for gene therapy



Infect dividing and non
-
dividing cells


Source: http://ksj.mit.edu/tracker/2009/05/ap
-
phil
-
inquirer
-
etc
-
new
-
tactic
-
immunity

Uses of
Adeno
-
associated
Viruses



Used in tissue engineering studies



Treat skin burns, excision and incision
wounds



Vectors are stable in different tissues

Retroviruses

Source: Warnock,
Daigre

and Al
-
Rubeai
, 2011.


Capsid surrounded by lipid
bilayer


contains surface and
transmembrane

glycoproteins

encoded by retrovirus


Genome contains linear,
ssRNA


3 major genes: gag,
pol

and
env
,
encode for proteins responsible for
viral integration, replication and
encapsulation


Only infect dividing cells


Life Cycle of Retroviruses



Bind to specific host cell receptors



Viral envelope fuses with host cell
membrane, releases viral contents



Reverse transcription occurs,
produces
dsDNA

and integrates into
host cell genome (provirus)


Transcription occurs, provirus
transcribed into mRNA, coded for
viral proteins



Reassemble in cytoplasm and exit
cell by budding

Source:
Ratcliffe
, 2012. IMM250.

Uses of Retroviruses



M
ostly in tissue repair and engineering



Construction of retroviral vectors require the
removal of gag,
pol

and
env

genes, allowing the
gene of interest to be inserted into the viral
genome



Retroviral vectors have be used in attempt to
treat X
-
linked severe combined immunodeficiency
(X
-
SCID), but adverse effects have been reported
in
clincial

trials

Gene Therapy

Non
-
viral Methods



Low
toxicity, lack of immune response and can be
easily prepared



Not
as efficient as viral methods due to:


-

low and transient
transgene

expression



-

limited ability to bind to surfaces of target
cells


-

ineffective in transporting the DNA through

the
nuclear membrane to the nucleus



Injection of naked plasmid DNA locally or systemically by a
needle injection (ex. intramuscular)



An injection with physical or electrical forces applied


-

Create pores in the plasma membrane



Electroporation



transport DNA directly across the
membrane with the application of an electric current

Source: http://spectrum.ieee.org/biomedical/devices/electroporation
-
knife
-
for
-
cancer

Source: http://9e.devbio.com/article.php?ch=2&id=290

Physical Approaches



Synthetic or natural compounds are used to form
electrostatic interactions with plasmid DNA



Due to the negative nature of DNA, positively
charged compounds such as cationic lipids and
polymers form
lipoplex

and
polyplex
, respectively



Lipoplex

and
polyplex

enter the cell by
endocytosis

and DNA release is based on different cellular
conditions that affect the
endosomes

Source: http://mgl.scripps.edu/people/goodsell/mgs_art/mgs_art2/gaber2.html

Chemical
Approaches

Summary


DNA
-
based diagnostics use DNA analysis to detect nucleic acids associated with a
disease


DNA Techniques for DNA diagnosis:


Polymerase Chain Reaction (PCR)


Hybridization


Sequencing


Gene therapy involves viral and non
-
viral methods


Somatic cell gene therapy


G
erm line gene therapy


M
odifies germ cells and thus descendants inherit this modification as well


Non
-
viral methods are not as effective as viral methods


P
rovides alternate mode of delivery of biological products to the body


Adenoviruses can be used as viral vectors to treat cancer


Adeno
-
associated viruses carry
ssDNA

and retroviruses carry
ssRNA


both can be used as viral vectors for gene therapy

References



Bank, A. (1996). Human somatic cell gene therapy.
BioEssays
,
18
(12), 999

1007.


Ferrari, M.,
Cremonesi
, L.,
Carrera
, P., &
Bonini
, P. A. (1996). Molecular diagnosis of genetic
diseases.

Clinical Biochemistry
,

29
(3), 201

208. doi:10.1016/0009
-
9120(95)02022
-
E


Grieger
, J.C., &
Samulski
, R.J. (2012).
Adeno
-
associated virus
vectorology
, manufacturing and clinical
applications.
Methods in
Enzymology
, 507, 229


254.


Ledley
, F. D. (1994). Development in Somatic Gene Therapy,
3
(9), 913

921.


Mountain, A. (2000). Gene therapy: the first decade.
Trends in Biotechnology
,
18
(3), 119

128.


Limberis
, M.P. (2012). Phoenix rising: gene therapy makes a comeback.
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Biochim
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Biophys
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44(8), 632


640.


Matthews, J. Nuclear receptor I. PCL302, (October 31, 2011).


Matthews, J. Nuclear receptor III. PCL302, (November 08, 2011).


M. T.
Tammi
,

The Principles of Shotgun Sequencing and Automates Fragment Assembly
, Center for
Genomics and Bioinformatics,
Karolinska

Institute, Stockholm, Sweden, 2003.


Muldrew
, K. (2009). Molecular diagnostics of infectious diseases.

Current Opinion in Pediatrics
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21
(1), 102
-
111.


Ratcliffe
, M. Being a B cell: how antibodies neutralize pathogens. IMM250, (March 6, 2012).


Roth, J.A. (2006). Adenovirus p53 gene therapy.
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Opin
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61.


Salvi
, M. (2011). Shaping Individuality: Human Inheritable Germ Line Gene Modification.
Theoretical
Medicine and Bioethics
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542.


Sheridan, C. (2011). Gene therapy finds its niche.
Nature Biotechnology
, 29(2), 121


128.


Warnock, J.N.,
Daigre
, C., & Al
-
Rubeai
, M. (2011). Introduction to viral vectors.
Methods in Molecular
Biology
, 737, 1


25.


Watcharanurak
, K., Nishikawa, M., Takahashi, Y., &
Takakura
, Y. (2012). Controlling the kinetics of
interferon
transgene

expression for improved gene therapy.
Journal of Drug
Targetting
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6.


Weber, J. L., & Myers, E. W. (1997). Human Whole
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Genome Shotgun Sequencing.

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409.