Biology Investigatory
Biology Investigatory
Project
Project
Gene Therapy
Gene Therapy
Name: Ragib Javed
Name: Ragib Javed
Class:
Class:
Secton:
Secton:
Certificate
Certificate
Y
Y
ear:
ear: 2013-1
2013-1
4
4
This is to certify that OV Shashank a student of
This is to certify that OV Shashank a student of
RN Podar Schoo! of cass "##- $! Ro
RN Podar Schoo! of cass "##- $! Ro
No:
No:
!
!
has co%&eted his fu se%ester
has co%&eted his fu se%ester
&ro'ect
&ro'ect in the fu
in the fu
(i%ent o
(i%ent of curricuu% )
f curricuu% )
#ndia
#ndia
Senior Secondary *+a%ination
Senior Secondary *+a%ination
The &ro'ect ,ork entited
The &ro'ect ,ork entited
Gene Therapy
Gene Therapy
! is the
! is the
oriina ,ork done .y hi% durin his a.o/e
oriina ,ork done .y hi% durin his a.o/e
fu se%e
fu se%e
ster &r
ster &r
o'ect
o'ect
$ate:
$ate:
#nterna
#nterna
*+a%iner
*+a%iner
Princi&a
Princi&a
*+terna
*+terna
*+a%iner
*+a%iner
Schoo
Schoo
Sta%&
Sta%&
Acknowledgement
# take this o&&ortunity to e+&ress %y sincere ratitude
to the honoura.e Princi&a rs )/nita ir of RN
Podar Schoo for her dee& interest and uidance
&ro/ided to %e durin the course
# a% %ost ratefu to our iooy teacher rs
Pad%a/athi for her reat he& in the co%&etion of
this &ro'ect
Student’s Signature
__________________________
Introduction
Gene Therapy
Targets
Types of gene therapy
Isolation of gene
Gene Targeting
Gene Delivery
Case Study – Cystic Fibrosis
The Disease
Is it a good Target
Choosing Vectors
History
Challenges
Ethical Issues
ecent !pco"ing
CIS#
Conclusion
$ibliography
%ebsites
$oo&s
Introduction
Diseases
The term disease broadly refers to any condition that impairs normal
function, and is therefore associated with dysfunction of normal
homeostasis. When the functioning of one or more organs or systems of the body is adversely affected, characterised by various signs and
symptoms, we say that we are not healthy, i.e., we have a disease.
Health can be defined as a state of complete physical, mental and social well-being. When people are healthy, they are more efficient at work. This increases productivity and brings economic prosperity. Health also increases longevity of people and reduces infant and maternal mortality. In humans, disease is often used more broadly to refer to any condition
that causes pain, dysfunction, distress, social problems, or death to the person afflicted, or similar problems for those in contact with the person. In this broader sense, it sometimes
includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behaviours, and atypical variations of structure and function, while in other contets and for other purposes these may be considered distinguishable categories.
!ased on the cause diseases can be broadly classified as"
Infections
These are diseases caused due to invasion of a foreign parasitic organism. They are temporary because the immune system of organisms can fight such pathogens #disease causing organisms$ to a certain etent hence helping in prevention of the disease. The immune system can also be aided
with the use of several drugs. %part from easy treatment they can also be easily prevented.
Lifestyle Diseases
&ifestyle diseases #also sometimes called diseases of
appear to increase in fre(uency as countries become more industriali'ed and people live longer. )iet and lifestyle are major factors thought to
influence susceptibility to many diseases. )rug abuse, tobacco smoking, and alcohol drinking, as well as a lack of eercise may also increase the risk of developing certain diseases, especially later in life. These
diseases can be prevented completely by living a healthy lifestyle.
*ertain diseases, such as diabetes, dental caries and asthma, appear at greater rates in young populations living in the +western+ way their
increased incidence is not related to age, so the terms cannot accurately be used interchangeably for all diseases.
Genetic Disorders
% genetic disorder is an illness caused by one or more abnormalities in the genome, especially a condition that is present from birth
#congenital$. They are medical disorders related to gene mutation.
enetic disorders are heritable, and are passed down from the parents genes. /ther defects may be caused by new mutations or changes to
the )0%. In such cases, the defect will only be heritable if it occurs in the germ line. The
same disease, such as some forms of cancer, may be caused by an inherited genetic
condition in some people, by new mutations in other people, and by non-genetic causes in still other people.
These diseases are totally random and difficult to prevent as they are not caused by eternal agents. %lso as their root cause lies in the genome of the organism their cure was thought to be impossible until the
breakthrough research unlocking the secrets of )0% leading to the development of biotechnology and hence gene therapy.
Gene Therapy
%e can thin& of a "edical condition or illness as a 'bro&en (indo()' *any "edical conditions result fro" fla(s+ or "utations+ in one or "ore of a person,s genes) *utations cause the protein encoded by that gene to "alfunction) %hen a protein
"alfunctions+ cells that rely on that protein,s function can,t behave
nor"ally+ causing proble"s for (hole tissues or organs) *edical conditions related to gene "utations are called genetic disorders)
So+ if a fla(ed gene caused our 'bro&en (indo(+' can (e 'fi-' it. %hat are our options.
/) Stay silent0 ignore the genetic disorder and nothing gets fi-ed)
1) Try to treat the disorder (ith drugs or other
approaches0 depending on the disorder+ treat"ent "ay or "ay not be a good long2ter" solution)
3) #ut in a nor"al+ functioning copy of the gene0 if you
can do this+ it "ay solve the proble"4
If it is successful+ gene therapy provides a (ay to fi- a
proble" at its source) 5dding a corrected copy of the gene "ay help the affected cells+ tissues and organs (or&
approaches+ (hich "ay treat the proble"+ but (hich do not repair the underlying genetic fla()
Gene therapy is the therapeutic delivery of nucleic acid polymers into a patients cells as a drug to treat disease. The first therapeutic use of gene transfer as well as the first direct insertion of human )0% into the nuclear genome was performed by 1rench %nderson in a trial starting in 2eptember 3445.
Targets for Gene Therapy
$ut no( a 6uestion arises+ (hich disorders or diseases can (e target using gene therapy. *any disorders or
"edical conditions "ight be treated using gene therapy+ but others "ay not be suitable for this approach) For a disease to be targeted by gene therapy it "ust satisfy the follo(ing conditions0
/) The condition "ust result fro" "utations in one or "ore genes)
1) To treat a genetic fla(+ the &no(ledge of (hich
gene7s8 to pursue is absolutely necessary) 5 D95 copy of that gene available in the laboratory) The best
candidates for gene therapy are the so2called 'single2 gene' disorders 2 (hich are caused by "utations in only one gene)
3) To design the best possible approach+ &no(ledge about ho( the gene factors into the disorder is re6uired) For e-a"ple0
%hich tissues are affected.
%hat role does the protein encoded by the gene play (ithin the cells of that tissue.
E-actly ho( do "utations in the gene affect the protein,s function.
:) 5dding a nor"al copy of the gene should fi- the proble" in the affected tissue) This "ay see" li&e obvious+ but it,s not) %hat if the "utated gene
encodes a protein that prevents the nor"al protein fro" doing its ;ob. *utated genes that function this (ay are called do"inant negative and adding bac& the nor"al protein (on,t fi- the proble")
<) The gene delivery to cells of the affected tissue "ust be possible) It depends on0
Ho( accessible is the tissue. Is it fairly easy 7s&in+ blood or lungs8+ or "ore difficult to reach 7internal organs8.
%hat is the best "ode of delivery.
The techni6ues of biotechnology have "ade it possible to isolate the re6uired gene in the laboratory and also deliver the gene)
Types of Gene
Cell types
Gene therapy may be classifed into two types:
Somatic
In so"atic cell gene therapy 7SCGT8+ the therapeutic genes are transferred into any cell other than
a ga"ete+ ger" cell+ ga"etocyte or undifferentiated ste" cell) 5ny such "odifications affect the individual patient only+ and are not inherited by offspring) So"atic gene therapy represents "ainstrea" basic and clinical
research+ in (hich therapeutic D95 7either integrated in the geno"e or as an e-ternal episo"e or plas"id8 is used to treat disease)
=ver >?? clinical trials utili@ing SCGT are under(ay in the !S) *ost focus on severe genetic disorders+
including i""unodeficiencies+ hae"ophilia+ thalassae"ia and cystic fibrosis) Such single gene disorders are good candidates for so"atic cell therapy) The co"plete
correction of a genetic disorder or the replace"ent of
"ultiple genes is not yet possible) =nly a fe( of the trials are in the advanced stages)
Germline
In ger"line gene therapy 7GGT8+ ger"
cells 7sper" or eggs8 are "odified by the introduction of functional genes into their geno"es) *odifying a ger" cell causes all the organis",s cells to contain the "odified
gene) The change is therefore heritable and passed on to later generations) 5ustralia+ Canada+ Ger"any+ Israel+
S(it@erland and the 9etherlands prohibit GGT for
application in hu"an beings+ for technical and ethical reasons+ including insufficient &no(ledge about possible ris&s to future generations and higher ris&s versus
SCGT) The !S has no federal controls specifically addressing hu"an genetic "odification 7beyond FD5 regulations for therapies in general8)
Isolation of Gene of Interest
The first step is to find and isolate the gene that (ill be inserted into the genetically "odified organis") Finding the right gene to insert usually dra(s on years of
scientific research into the identity and function of useful genes) =nce that is &no(n the D95 needs to be cut at
specific locations to isolate the gene of interest) This can be done by using restriction en@y"es also &no(n as
"olecular scissors (hich cut D95 at specific sites
containing palindro"ic D95 se6uences) $ut in order to cut the D95 (ith restriction en@y"es+ it needs to be in pure for"+ free fro" other "acro2"olecules)
!ntil the early /AB?s D95 (as the "ost difficult cellular "olecule for the bioche"ist to analyse)
Enor"ously long and che"ically "onotonous+ the string of nucleotides that for"s the genetic "aterial of an
organis" could be e-a"ined only indirectly+
by protein or 95 se6uencing or by genetic analysis)
Today the situation has changed entirely) Fro" being the "ost difficult "acro"olecule of the cell to analyse+ D95 has beco"e the easiest)
Isolation of DNA
Since the D95 is enclosed (ithin the "e"branes+ (e have to brea& the cell open to release D95
along (ith other "acro"olecules such as 95+ proteins+ polysaccharides and also lipids) This can be achieved by treating the bacterial cellsplant or ani"al tissue (ith en@y"es such as lyso@y"e 7bacteria8+ cellulase 7plant cells8+ chitinase 7fungus8) Genes are located on long "olecules of D95
intert(ined (ith proteins such as histones) The 95 can be re"oved by treat"ent (ith ribonuclease (hereas
proteins can be re"oved by treat"ent (ith protease) =ther "olecules can be re"oved by appropriate
treat"ents and purified D95 ulti"ately precipitates out after the addition of chilled ethanol) This can be seen as collection of fine threads in the suspension)
Cutting of DNA
estriction en@y"e digestions are perfor"ed by incubating purified D95 "olecules (ith the
restriction en@y"e+ at the opti"al conditions for that specific
en@y"e) The cutting of D95 by restriction endonucleases results
in the frag"ents of D95) These frag"ents can be
separated by a techni6ue &no(n as gel electrophoresis) Since D95 frag"ents are negatively charged "olecules they can be separated by forcing the" to "ove to(ards the anode under an electric field through a
for the re6uired gene and then it is cut out fro" the
agarose gel and e-tracted fro" the gel piece) This step is &no(n as elution)
ultiplication of Gene !PC"#
#C or poly"erase chain reaction is then used to create "ultiple copies of the gene of interest) In this reaction+ "ultiple copies of the gene 7or D958 of interest is
synthesised in vitro using t(o sets of pri"ers 7s"all che"ically synthesised oligonucleotides that are
co"ple"entary to the regions of D958 and the en@y"e D95 poly"erase) The en@y"e e-tends the pri"ers using the nucleotides provided in the reaction and the geno"ic D95 as te"plate) If the process of replication of D95 is repeated "any ti"es+ the seg"ent of D95 can be
a"plified to appro-i"ately billion ti"es+ i)e)+ / billion copies are "ade)
#C is a (onderful technology for a"plifying D95) It allo(s you to ta&e a specific region of D95 on the
chro"oso"e and through the use of pri"ers+ copy bac& and forth+ only a particular desired seg"ent+ "a&ing t(o+ then four+ then eight+ then si-teen+ and so on+ up to
"illions of copies) It is possible to start fro" the D95 seg"ent of a single cell and produce enough of it for use in DNA typing or $ingerprinting%
#C relies on the ability of D952copying en@y"es to re"ain stable at high te"peratures) The process (as spar&ed by a high te"perature bacteriu" 7Ther"us a6uaticus8 inhabiting the hot springs of ello(stone 9ational #ar&.
Gene Targeting
Gene targeting 7also+ replace"ent strategy based on ho"ologous reco"bination8 is a genetic techni6ue that uses ho"ologous reco"bination to change
an endogenous gene) The "ethod can be used to delete a gene+ re"ove e-ons+ add a gene+ and introduce point
"utations) Gene targeting can be per"anent or conditional) Conditions can be a specific ti"e
during develop"ent life of the organis" or li"itation to a specific tissue)
Gene delivery is one of the biggest challenges in
the field of gene therapy)
Gene Delivery includes&
/) T5GETI9G the right cells)
1) 5CTIV5TI9G the gene) 5 gene,s ;ourney is not over (hen it enters the cell) It "ust go to the cell,s nucleus and be 'turned on+' "eaning that its transcription and translation are activated to produce the protein product encoded by the gene) For gene delivery to be successful+ the protein that is produced "ust function properly)
3) I9TEG5TI9G the gene in the cells) The gene "ust stay put and continue (or&ing in the target cells) If so+ it "ust be ensured that the gene integrates into+ or beco"es part of the host cell,s genetic "aterial+ or that the gene finds another (ay to survive in the nucleus (ithout being
re;ected)
:) 5V=IDI9G har"ful side effects) 5nyti"e an unfa"iliar biological substance is introduced into the body+ there is a ris& that it (ill be to-ic or that the body (ill "ount an
i""une response against it) If the body develops i""unity against a specific gene delivery vehicle+ future rounds of the therapy (ill be ineffective) For gene delivery to be successful+ foreign D95 "ust survive long enough in the host cell to integrate into its geno"e) This re6uires foreign D95 to be synthesi@ed as part of a vector+ (hich is
designed to enter the desired host cell and deliver
the transgene to that cell,s geno"e )Vectors utili@ed as the "ethod for gene delivery can be divi ded into t(o categories+ non2viral and viral)
Choosing the
Best
'ector
There is no 'perfect vector' that can treat every disorder) i&e any type of "edical treat"ent+ a gene therapy vector "ust be custo"i@ed to address the uni6ue features of the disorder) %e have learnt the lesson+ of transferring genes into plants and ani"als fro" bacteria and viruses+ (hich have &no(n this for ages – ho( to deliver genes to
transfor" eu&aryotic cells and force the" to do (hat the bacteria or viruses (ant)
Gene therapy
utili@es the delivery of D95 into cells+ (hich can be acco"plished by several "ethods+su""ari@ed belo() The t(o "a;or classes of "ethods are those that use reco"binant viruses 7so"eti"es called biological nanoparticles or viral vectors8 and those that use na&ed D95 or D95 co"ple-es 7non2viral "ethods8)
#art of the challenge in gene therapy is choosing the "ost suitable vector for treating the disorder) So"e vectors
co""only used are0
Viruses
!sually (hen (e thin& of viruses+ (e thin& of the"
HIV5IDS) %hen faced (ith the proble" of gene delivery+ scientists loo&ed to viruses) %hy reinvent the (heel if there,s a perfectly good one out there. If (e can "odify viruses to deliver genes (ithout "a&ing people sic&+ (e "ay have a good set of gene therapy tools)
5ll viruses bind to their hosts and introduce their genetic "aterial into the host cell as part of their replication
cycle) This genetic "aterial contains basic ,instructions, of ho( to produce "ore copies of these viruses+ hac&ing the body,s nor"al production "achinery to serve the
needs of the virus)
The host cell (ill carry out these instructions and produce additional copies of the virus+ leading to "ore and "ore cells beco"ing infected) So"e types of viruses insert their geno"e into the host,s cytoplas"+ but do not actually enter the cell) =thers penetrate the cell
"e"brane disguised as protein "olecules and enter the cell)
General advantages of viral vectors0 2They,re very good at targeting and entering cells)
2So"e viral vectors "ight be engineered to target specific types of cells)
2They can be "odified so that they can,t replicate and destroy the cell)
General dra(bac&s of viral vectors0
5 virus can,t 'e-pand' to fit a piece of genetic "aterial larger than it is naturally built to carry) Therefore+ so"e genes "ay be too big to fit into a certain type of virus)
Viruses can cause i""une responses in patients+ resulting in t(o potential outco"es0
#atients "ay get sic&)
5 patient,s i""unity to a virus "ay prevent hi" fro" responding to repeated treat"ents)
Ho(ever+ "odern viral vectors have been engineered
(ithout "ost of the proteins that (ould cause an i""une response)
NonViral Vectors
5lthough viruses can effectively deliver genetic "aterial into a patient,s cells+ they do have so"e li"itations) It is so"eti"es "ore efficient to deliver a gene using a non2 viral vector+ (hich has fe(er si@e constraints and (hich (on,t generate an i""une response)
9on2viral vectors are typically circular D95 "olecules+ also &no(n as plas"ids) In nature+ bacteria use plas"ids to transfer genes fro" cell to cell)
Scientists use bacteria and plas"ids to easily and
efficiently store and replicate genes of interest fro" any organis")
Vectors used at present+ are engineered in such a (ay that they help easy lin&ing of foreign D95 and selection of
reco"binants fro" non2reco"binants)
These are not the only way to introduce alien !NA into host cells) In a "ethod &no(n as "icro2in;ection+ reco"binant D95 is
directly in;ected into the nucleus of an ani"al cell) In another "ethod+ suitable for plants+ cells are bo"barded (ith high velocity "icro2particles of gold or tungsten
coated (ith D95 in a "ethod &no(n as biolistics or gene gun)
Delivery to speci$ic tissues
Delivering genes to specific tissues (ithin a patient,s
body can be very difficult) Delivering genes into a group of cells in a patient,s body can be done in one of t(o (ays) The first (ay is to in;ect the vector into the body and
specifically target affected cells) This is called an in vivo approach) The second (ay+ called e- vivo + is to deliver the gene to cells (hile they,re outside the body by0
Isolating the desired cells fro" the body)
Culturing the cells in a #etri dish in the laboratory)
Delivering the genes to the cells 7using one of the
vector options described on this page8+ activating the"+ and "a&ing sure that the cells integrate the" properly)
Case Study "
The Disease ( A Genetic Disorder
Cystic fibrosis 7CF8+ also &no(n as "ucoviscidosis+ is an autoso"al
recessive genetic disorder that affects "ost critically the lungs+ and also the pancreas+ liver+ and intestine) It is
characteri@ed by abnor"al transport of chloride and sodiu" across an epitheliu"+ leading to thic&+ viscous secretions+
preventing the cilia fro" clearing debris (hich cause sy"pto"s such as coughing+ poor digestion and increased vulnerability to infection)
CF is caused by a "utation in the gene for the protein cystic fibrosis trans"e"brane conductance regulator 7CFT8 gene on chro"oso"e B) *ost co""only+ the "utation in the CFT gene is a three2 base2pair deletion) This protein is re6uired
to regulate the co"ponents of s(eat+
digestive fluids+ and "ucus) CFT regulates the "ove"ent of chloride and sodiu" ions across epithelial "e"branes+ such as the alveolar epithelia located in the lungs) Since all of the cells of a CF patient have the defective protein+ large 6uantities of thic&+ stic&y "ucus build up throughout the lungs and other organs) This results in the severity of sy"pto"s seen in CF patients)
To chec& this so"e 6uestions "ust be ans(ered0
!oes the condition result rom mutation$ es)
Is the biology o the disorder known$ es)
%ill adding a normal copy o the gene fi& the problem in the aected tissue$ es) %hile the "utated CFT gene encodes a non2functional ion channel protein+ it (ill not prevent a nor"al CFT channel protein fro"
(or&ing properly) Therefore+ adding a nor"al copy of the CFT gene should fi- the proble"
Is it easible to deli'er the gene to the cells o the aected tissue$ es+ in part) Treating the lungs of patients (ith CF "ight be feasible+ since the lung surfaces are e-posed to the air and so"e(hat easy to reach) $ecause the digestive syste" is less accessible+ ho(ever+ it "ight be a "ore difficult region to treat)
Hence (e can conclude that it is a perfect disease to be treated by gene therapy)
Choosing vectors
The vectors that are "ost suitable for gene therapy are0
"etrovirus
etroviruses are enveloped viruses that replicate in a host cell
through the process of reverse transcription) It is a single2stranded 95 virus that stores its nucleic acid in the for" of an "95
geno"e targets) =nce inside the host cell cytoplas" the virus uses its o(n reverse transcriptase en@y"e to produce D95 fro" its 95 geno"e+ the reverse of the usual pattern+ thus retro 7bac&(ards8) This ne( D95 is then incorporated into the host cell geno"e by an integrase en@y"e+ at (hich point the retroviral D95 is referred to as a provirus) The host cell then treats the viral D95 as part of its o(n geno"e+ translating and transcribing the viral genes along (ith the cell,s o(n genes+ producing the proteins re6uired to asse"ble ne( copies of the virus)
=ne dra(bac& of retroviruses+ such as the *oloney retrovirus+ involves the re6uire"ent for cells to be actively dividing for
transduction) 5s a result+ cells such as neurons are very resistant to infection and transduction by retroviruses)
$ut the air(ay cells (hich are affected by the disease cystic
fibrosis and "ust be targeted divide infre6uently) Hence etrovirus is not a suitable vector for this disease)
Adenovirus
5denoviruses 7"e"bers of the fa"ily 5denoviridae8 are "ediu"2si@ed 7A?– /?? n"8+ nonenveloped 7(ithout an outer lipid bilayer8 viruses (ith anicosahedral nucleocapsid
containing a double stranded D95 geno"e)
They have a broad range of vertebrate
hosts and have been found to cause a (ide range of illnesses+ fro" "ild respiratory infections in young children to life2threatening
"ulti2organ disease in people (ith a (ea&ened i""une syste") $ut they can causeCinduce an i""une response in the hu"an body hence not suitable for gene delivery)
+erpes ,i-ple. 'irus
Herpes si"ple- viruses+ also &no(n as Hu"an herpes virus+ are "e"bers of the herpes virus fa"ily+
Herpesviridae+ that infect hu"ans) They can be spread (hen an infected person is producing and shedding the
virus) Herpes Si"ple- can be spread through contact (ith saliva+ such as sharing drin&s)
$ut these viruses only affect the cells of the nervous syste" and cannot infect the air(ay cells and hence not suitable)
Adeno/Associated 'iruses
5deno2associated virus 755V8 is a s"all virus (hich infects hu"ans and so"e other pri"ate species) 55V is not currently &no(n to
cause disease and conse6uently the virus causes a very
"ild i""une response)55V can infect both dividing and 6uiescent cells and persist in an e-tra chro"oso"al state (ithout integrating into the geno"e of the host cell) Despite its fe( disadvantages
these features "a&e 55V a very attractive candidate for creating viral vectors for gene therapy+ and for the creation of isogenic hu"an disease "odels
Hence it is the best choice for gene delivery in the case of Cysc Fibrosis.
+istory of Cystic )i0rosis Gene Therapy
Gene therapy for cystic fibrosis began in /AA?+ (hen scientists successfully corrected faulty cystic fibrosis trans"e"brane
conductance regulator 7CFT8 genes) They did this by adding nor"al copies of the gene to laboratory cell cultures)
1223
In /AA3+ the first e-peri"ental CF gene therapy treat"ent (as given to a patient (ith cystic fibrosis) esearchers "odified a co""on cold 5denovirus to act as a delivery vehicle by carrying nor"al genes to the CFT cells in the nasal passages) esearchers chose nasal passages as the site of delivery because they are easier to access and "easure gene activity than the lung air(ay) ater trials delivered the vector to patients lung air(ays)
In the earlier trials+ it had loo&ed li&e the virus had entered cells and that the CTF gene (as (or&ing) $ut later trials (ith different
patients sho(ed levels of VFT gene activity that (ere too lo( to "a&e any difference) esearchers ca"e to thin& that the adenovirus cant easily enter air(ay cells+ especially in the lo( doses that (ere being given) In the earlier trials+ they speculated+ gene activity
resulted fro" the da"age to the cells during delivery allo(ing the virus to enter easily)
Hence (hen higher doses of the virus (ere tried+ the i""une syste" of the patients started "ounting i""une responses and
fighting off the virus) This caused a bloc&age in the trials until /AA)
1224
Trials using 5deno2associated virus to deliver the CTF gene began in /AA) !nli&e the adenovirus+ the 5deno2associated virus caused no i""une response or adverse side effects in patients)
$ut unli&e the researchers predictions+ the adeno2associated virus did not enter cells efficiently and integrate into calls geno"ic D95) They
produced only lo( and fleeting a"ounts of CFT gene activity)
esearchers are still (or&ing to figure out (hat caused the viruses to fail) $ut because it is safe+ the virus – as (e predicted earlier – holds pro"ise for being a good (ay to deliver the CFT gene to patients air(ay cells) $ut researchers need to learn "ore about ho( the virus infects cells in order or "a&e it an effective delivery "ethod)
Challenges
So"e the factors that have &ept gene therapy fro" beco"ing an effective treat"ent for genetic diseases are0
Shortli'ed nature o gene therapy $efore gene therapy can
beco"e a per"anent cure for any condition+ the therapeutic D95 introduced into target cells "ust re"ain functional and the cells containing the therapeutic D95 "ust be long2lived
and stable) #roble"s (ith integrating therapeutic D95 into the geno"e and the rapidly dividing nature of "any cells prevent gene therapy fro" achieving any long2ter" benefits) #atients (ill have to undergo "ultiple rounds of gene therapy)
Immune response 5nyti"e a foreign ob;ect is introduced into
hu"an tissues+ the i""une syste" is designed to attac& the invader) The ris& of sti"ulating the i""une syste" in a (ay that reduces gene therapy effectiveness is al(ays a potential ris&) Further"ore+ the i""une syste",s enhanced response to invaders it has seen before "a&es it difficult for gene therapy to be repeated in patients)
(roblems with 'iral 'ectors Viruses+ (hile the carrier of choice
in "ost gene therapy studies+ present a variety of potential proble"s to the patient 22to-icity+ i""une and infla""atory responses+ and gene control and targeting issues) In addition+ there is al(ays the fear that the viral vector+ once inside the patient+ "ay recover its ability to cause disease)
)ultigene disorders Conditions or disorders that arise fro"
"utations in a single gene are the best candidates for gene therapy) !nfortunately+ so"e the "ost co""only occurring disorders+ such as heart disease+ high blood pressure+
5l@hei"er,s disease+ arthritis+ and diabetes+ are caused by the co"bined effects of variations in "any genes) *ultigene or "ultifactorial disorders such as these (ould be especially difficult to treat effectively using gene therapy)
Issues regarding
Gene Therapy
%hat are the possible i"plications of gene therapy research to
responsibility to e-plore the potential effects of gene therapy research on our lives so that (e can "a&e infor"ed decisions) For each ne( application of gene therapy research+ (e "ust consider0
%hat are the benefits.
%hat are the ris&s.
%ho" (ill the technology help. %ho (ill it potentially hurt.
%hat does gene therapy "ean for us.
There are several types of issues to consider as (e thin& about gene therapy0
Ethical issues as& us to consider the potential "oral outco"es
of gene therapy research)
egal issues re6uire researchers and the public to help
policy"a&ers decide (hether and ho( gene therapy research should be regulated by the govern"ent)
Social issues involve the i"pact of gene therapy research on
society as a (hole)
So"e 6uestions to ponder
%hen should gene therapy be used$ Should it be used to treat critically ill patients. Should it be used to treat babies and children.
%hat eect would gene therapy ha'e on uture generations i germline
*reproducti'e+ cells were genetically altered$ Ho( "ight this alteration affect hu"an variation.
%ho should decide what are ,good, or ,bad, uses o genetic
modifications$ Ho( do you define 'nor"al' (ith regard to hu"an beings.
%hat i we could alter human traits not associated with disease$ %ould it be o&ay to use gene therapy to i"prove or enhance a person,s genetic profile.
%ho will ha'e access to gene therapy- treatments and longterm ollow ups$ %ill gene therapy and genetic enhance"ents create an advantage for those (ho can afford it.
.ecent /pcoming
C"I,P"
CIS# stands for clustered regularly interspaced short
palindro"ic repeats) These 95 se6uences serve an i""une
function in archaea and bacteria+ but in the last year or so+
scientists have sei@ed upon the" to re(rite genes) The 95
se6uence serves as a guide to target a D95 se6uence in+ say+ a @ygote or a ste" cell) The guide se6uence leads an en@y"e+
CasA+ to the D95 of interest) CasA can cut the double strand+ nic& it+ or even &noc& do(n gene e-pression) 5fter CasA in;ures the D95+ repair syste"s fi- the se6uence 2 or ne( se6uences can be
inserted)
It isn,t the first or only "ethod of gene repair therapy thats been developed+ but the CIS# technology+ says a"esar+ is so special because+ unli&e previous "ethods (hich (ere "ore laborious and could only target one &ind of cell in the body+ it appears to be a 'one si@e fits all delivery'+ adaptable for different tissues) The procedure also see"s relatively si"ple to perfor")
E-citing as the develop"ent "ay be+ CIS# (ont be delivering instant cures ;ust yet)
a"esar says+ fro" his initial i"pressions of the literature+ that it (ould see" that localised+ accessible abnor"al tissue 7as in the retina or s&in8 could be targeted "ore easily)
Conditions affecting the body "ore syste"ically+ ho(ever+ such as certain develop"ental syndro"es+ or central nervous syste"
disorders+ "ight be proble"atic in ter"s of getting the repair
technology into enough of the target cells in that tissue to "a&e an effective difference)
'It "ay also depend on the stage one atte"pts to carry out the therapy+ in ter"s of the patients age and level of advance"ent of the disease+' says a"esar)
Conclusion
5lthough early clinical failures led "any to dis"iss gene therapy as over2hyped+ clinical successes since 1??> have bolstered ne(
opti"is" in the pro"ise of gene therapy) These include successful treat"ent of patients (ith the retinal disease eber,s congenital a"aurosis+ 2lin&ed SCID+ 5D52SCID+ adrenoleu&odystrophy+ chronic ly"phocytic leu&ae"ia 7C8+acute ly"phocytic
leu&ae"ia 758+"ultiple "yelo"a+ hae"ophilia and #ar&inson,s disease) These recent clinical successes have led to a rene(ed interest in gene therapy+ (ith several articles in scientific and popular publications calling for continued invest"ent in the field)
0ibliography
5e0sites
h!p:""en#wi$ipedia#or%"wi$i"Gene&therapy
h!p:""learn#%ene(cs#)tah#ed)"content"tech"%enetherapy" h!p:""%hr#nlm#nih#%o*"handboo$"therapy" h!p:""cys(c+fbrosis#emedt*#com"cys(c+fbrosis"cys(c+fbrosis+%ene+therapy#html h!p:""en#wi$ipedia#or%
