Biology Investigatory

Project
Gene Therapy

Name: OV Shashank 
Class: XII – D
Roll No:
Certifcate
Year: 2013-14
This is to certify that OV Shashank a
student of RN Podar Schoo! of cass "##-
$! Ro No: ! has co%&eted his
fu se%ester &ro'ect in the fu(%ent of
curricuu% ) #ndia Senior Secondary
*+a%ination
The &ro'ect ,ork entited Gene
Therapy ! is the oriina ,ork done
.y hi% durin his a.o/e fu
se%ester &ro'ect
$ate:

1
 
  
#nterna *+a%iner
Princi&a

  
*+terna *+a%iner
Schoo 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 iooy teacher
rs Pad%a/athi for her reat he& in the
co%&etion of this &ro'ect

2
 Student’s Signature


 Table o Contents
 I
ntr
oduct
ion
 GeneTher
apy
T
arg
ets
I
sol
ati
onofgene
 GeneTar
get
ing
 GeneDel
i
ver
y
 Cas
eSt
udy–Cy
sti
cFi
bros
is
TheDi
sease

3
 I
sitagoodTar
get
 Choosi
ngVect
ors
 Hi
st
ory
 Chal
l
enges
 Et
hicalI
ssues
 RecentUpcomi
ng
 CRI
SPR
 Conc
lus
ion
 Bi
bli
ogr
aphy
 Websi
t
es
  
Book
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.


Based on the cause diseases can be broadly classified as:

In!ections
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 e"tent 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

"i!estyle Diseases
$ifestyle diseases also sometimes called diseases of longevity or diseases of
civili%ation interchangeably! are diseases that appear to increase in fre&uency as
countries become more industriali%ed and people live longer. They can
include #l%heimer's disease, asthma, and obesity. (iet and lifestyle are ma)or factors
thought to influence susceptibility to many diseases. (rug abuse, tobacco smoking,
and alcohol drinking, as well as a lack of e"ercise may also increase the risk of
developing certain diseases, especially later in life. These diseases can be
prevented completely by living a healthy lifestyle.

#enetic Disor$ers
 # 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 (#. n 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 e"ternal 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 (# leading to the development of biotechnology and hence gene therapy.

!
Gene Therapy
Wec ant hi nkofamedi calc ondit
ionor
i
llnes sasa" brok e
nwi nd ow."Man yme dica
l
condi tionsr esultfrom flaws ,ormut ations,i
n
on eormo reo fape rs
on 'sgene s.Mut ati
ons
caus et hepr ot
einenc odedb ythatgenet o
mal func tion.Whenapr oteinmal func ti
ons,
cellst hatr elyont hatprotein'sfunctionc an't
beha v enor mal ly,causingpr oblemsf or
whol et iss uesoror gans .Medi calcondi ti
ons
relatedt ogenemut at
ionsar ec all
edgenet i
c
disor der s.

So,ifaflawedgen
ecau
sedo
ur"
brok
enwi
ndo
w,"c
anwe"
fix"i
t
?Whata
reo
ur
opt
ions?

1. St
ays
il
ent
:ignor
ethegenet
i
cdi
sor
derandnot
hingget
sfix
ed.

2. T
rytotr
eatthedi
sorderwit
hdrugsoro
therappr
oaches:dependi
ngont
he
disor
der
,tr
eatmentma yormaynotbeagoodlong-
terms ol
uti
on.

3. Puti
nanormal
,func
tioni
ngc
opyoft
hegene:i
fyouc
andot
his
,itma
ysol
ve
theprobl
em!

I
fitissuccessful,genet
herapyprov
idesawayt ofixaproblem atit
ssour
ce.Addi
ng
ac orr
ect
edc op yofthegenema yhelptheaffect
edcell
s,t
i
ss uesandorganswork
properl
y.Genet herapydi
ffersfr
om tradi
ti
onaldr
ug-
basedappr oac
hes,whic
hma y
tr
eattheproblem,butwhi c
hdonotr epairt
heunder
lyi
nggeneticflaw.

%argets !or #ene %hera&y

Butnow aques
tionar
ises
,whi
chdis
or der
sordi
seasescanwetar
getusi
nggene
t
herap
y?Man ydisor
dersormedi
calcondi
ti
onsmightbet
reat
edusi
nggenether
apy
,

"
butot
hersmaynotbesui
tabl
efort
hisappr
oach.Foradi
seas
etobet
arget
edby
genether
apyi
tmustsat
is
fythefol
l
owingcondi
ti
ons:

1. Thecondi
ti
onmustr
esul
tfr
om mut
ati
onsi
noneormor
egenes

2. Tot
reatageneticflaw,t
heknowl
edgeofwhi chgene(
s)t opursuei
s
absolut
elynecessar
y.Al
soaDNAc opyoft
hatgeneav
ailablei
nthe
l
aborat
ory.Thebes
tcandi
dat
esf
orgenet
her
apyaretheso-
call
ed"si
ngl
e-
gene"di
sorder
s-wh i
char
ecaus
edbymutat
i
onsinonlyonegene.

3. Todesi
gnthebestpossi
bleapproach,knowledgeabouthow t
hegene
fact
orsint
othedisor
derisrequi
red.Forexample:

 Whi
cht
i
ssue
sar
eaff
ect
ed?

 Whatr
oledoest
hepr
otei
nenc
odedbyt
hegenepl
aywi
t
hint
hec
ell
sof
t
hatt
i
s s
ue?

 Ex
act
lyhowdomut
ati
onsi
nthegeneaffec
tthepr
otei
n'
sfunc
ti
on?

4. Addinganormalcopyoft
hegeneshouldfixthepr
oblem i
ntheaffected
tissue.Thi
smayseem l
i
keobv
ious
,buti
t
'snot
.Whati
fthemut
atedgene
encodesapr ot
einthatpr
eventst
henor
malpr
otei
nfr
om doi
ngi
tsjob?Mutat
ed
genesthatfunc
tionthi
swa yarecal
l
eddomi
nantneg
ati
veandaddingbac
kthe
normalprot
einwon'tfixtheprobl
em.

5. Thegenedel
iver
ytocel
lsoft
heaffect
edt
issuemustbepossi
ble.I
t
dependson:

 Howacc
ess
ibl
eist
het
i
ssue?I
sitf
air
lyeas
y(sk
in,bl
oodorl
ungs)
,or
mor
edi
fficul
ttor
eac
h(i
nter
nal
organs
)?

 Whati
sthebes
tmodeofdel
i
ver
y?

Thet
echni
quesofbi
otechnol
ogyhavemadeitpos
sibl
etoi
sol
atet
her
equi
redgene
i
nthel
abor
ator
yandal s
odelivert
hegene.

Isolation o! #ene o! Interest

Thefir
stst
epistofindandis
olat
ethegenethatwil
lbeinser
tedi
ntot
hegenet
ic
all
y
modi
fiedorgani
sm.Fi
ndingt
heri
ghtgenetoinser
tusuall
ydrawsonyear
sof

#
sci
ent
ificresearchint
otheidentit
yandf unct
ionofusefulgenes .
 Oncet hatisknown
t
heDNAneedst obec utats peci
ficlocati
onstoisol
atethegeneofi nterest
.Thiscan
bedonebyus ingrest
ric
tionenzymesal soknownasmol ecularsci
ss orswhichc ut
DNAats pecificsi
tescontaini
ngpalindr
omicDNAs equences .Butinor dertocutthe
DNAwi t
hr est
ri
cti
onenz y
mes ,itneedstobei npureform,freefrom othermac ro-
molec
ules.

Isolation o! DN'
Si
ncetheDNAi
senc
lose
dwi th
inth eme mbrane s,weh av et
obr eakthecellopento
r
elea
seDNAalo
ngwit
hothermac romo l
eculess ucha sRNA,pr otei
ns,
poly
sac chari
desandal s
ol ipids.Thiscanbeac hievedby
tr
eati
ngt hebac t
eri
al cel
ls/plantoranimal t
i
s s
uewi t
h
enzymess uchasl ysozyme( bact
eri
a),cel
lulase(plant
cel
ls),chiti
nase(fungus ).Genesar elocat
edonl ong
molec ul
esofDNAi nt
ert
winedwi t
hprotei
nssuchas
hi
stone s.TheRNAc anber emovedb ytr
eat
mentwi t
h
r
ibon ucl
e asewhereasprotei
nsc anberemovedby
t
reatme ntwithprot
ease.Othermol e
culescanberemo ved
byappr opri
atetr
eatmentsandpur i
fiedDNAul t
imatel
y
preci
pit
atesoutaft
ert
headdit
ionofc hil
l
edethanol.Thiscanbes eenascol
lecti
onof
finethreadsinthesus
pensi
on.

C(tting o! DN'

Res tr
ict
ionenz ymedi gest
ionsar eper formedb y
i
nc ubati
ngpur ifiedDNAmol eculeswi tht he
restri
cti
onenz yme,att heopt i
mal condi t
i
onsf ort hat
spec i
ficenz yme.Thec utt
ingofDNAbyr estri
c t
ion
en donucleas esr es
ultsinthef r
ag men tsofDNA.
Th esefragme ntscanb es epa r
a tedb yat echnique
knownasge lelectrophoresis.Si nceDNA
f
r agment sar enegat i
velychargedmol eculest hey
ca nbes epa ratedb yforci
ngt hem t omo veto war ds
t
hea nodeu nderane l
ectr
icfi eldthrou ghamed ium/ mat
ri
x.Thes ep
arat
edban
dsof
DNAar eanal ys
edf ortherequi redgeneandt heni ti
scutoutfr
om t
heagar
osegel
ande xtractedf r
om thegel pi
ec e.Thisst epi sknownasel ut
ion.

$
)(lti&lication o! #ene *PCR+

PCRorpol y
mer asechainreacti
onisthenus edtoc r
eatemulti
plecopiesofthegene
ofinterest.I
nt hisreact
ion,multi
plecopiesofthegene( orDNA)ofi nt
eresti
s
synthes i
sedinv i
trousi
ngt wosetsofprimers( s
mal lchemical
lysynthesis
ed
ol
igon ucleot
ide sthatareco mpl
eme ntarytothere gio
nsofDNA)an dthee nzyme
DNAp olymer ase.Th ee nzymeex t
endst hepri
mer su si
ngthenuc l
eotidespr
o v
idedi
n
t
her eac ti
onandt hegenomi cDNAast emplate.Iftheprocessofrepli
cati
onofDNA
i
sr epe atedma nyt imes,thesegme ntofDNAc anb ea mpli
fiedtoa pproxi
mately
bi
ll
iont imes,i.e.,1bil
li
onc opi
esar emade.

#ene %argeting

Genedel
i
ver
yisoneoft
hebi
gges
tchal
l
engesi
nthefi
eldofgenet
her
apy
.

GeneDel
i
ver
yinc
ludes
:

1.TARGETI
NG t
her
ightcel
ls.

2.ACTI
VATI
NG t
hegene.
 
Agene'
sjour
neyi
snotov
erwheni
tent
erst
hec
ell
.It
mustgotothecel
l
'snuc l
eusandbe" t
urnedon,"meaningthati
tst
ranscr
ipt
i
onand
t
ransl
ati
onareact
ivat
edt opr
oduc et
heprotei
nproductencodedbythegene.For
genedeli
ver
ytobes uccessf
ul,t
heprot
einthati
sproducedmus tf
unct
ionproper
ly.

3.I
NTEGRATI
NG t
hegenei
nthecel
ls.
 Thegenemustst
ayputandcont
inue
worki
ngint
het ar
getcel
l
s.Ifso,i
tmustbeens uredt
hatt
hegenei
ntegr
atesi
nto,or
becomespartoft
hehostcell
'
sgeneti
cmat er
ial,ort
hatt
hegenefi
ndsanotherwayto
sur
v i
vei
nthenucleuswit
houtbei
ngrej
ected.

4.AVOI
DING har
mfulsi
deeffect
s. 
Any
timeanunf
ami
l
iarbi
ologi
cal
subs
tanc
eis
i
ntroduc
edi ntothebody,thereisaris
kthatitwil
l bet
oxi
corthatt
hebodywil
lmount
animmuner esponseagainstit
.Ift
hebodydev elopsi
mmuni t
yagainstas
pec
ific
genedeliv
er yvehic
le,f
utureroundsofthetherapywil
lbeineffect
ive.

%
Choosing the Best Vector

Thereisno" perfectvector"t
hatcantreatev erydisorder.Li
keanyt ypeofmedical
tr
eatment,agenet herapyv ect
ormus tbec ustomizedtoaddr es
st heuniquefeat
ures
ofthedis
order. Wehav elearntt
helesson,oft ransf
erri
nggenesi ntoplant
sand
animal
sfrom bac teri
aandv i
ruses,whichha vek nownt hisf
orages–ho wtodeli
ver
genestotransform euk aryot
i
ccellsandf or
c ethem todowhatt hebac teri
aorvir
uses
want.

Par
tofthechal
lengeingenether
apyi
schoos
ingthemos
tsui
t
abl
evec
torf
ort
reat
i
ng
t
hedisorder
.Somev ect
orscommonl
yusedare:

&iruses

Usual
lywhenwet hi
nkofv ir
uses,wethinkofthem c
aus i
ngdiseasessuchasthe
commo ncold,t
hefl u,andHI V/AI
DS.Whe nfa
ce dwit
hthepr o
blem ofgenedel
iver
y,
sci
ent
ist
slookedtov ir
uses.Whyr einv
entthewheelift
here'saperfect
lygoodone
outt
here?Ifwec anmodi fyvi
rusestodeli
vergeneswithoutmakingpeoplesi
ck,we
mayha veag oods etofgenetherapytool
s.

Gener
aladv
ant
agesofv
iralv
ect
ors
:

- They
'rev
erygoodatt
arget
i
ngandent
eri
ngc
ell
s.

 -
Somev
iral
vec
tor
smi
ghtbeengi
neer
edt
otar
get
s
pec
ifict
ypesofc
ell
s.

 - Theycanbemodi
fieds
othatt
heyc
an'
trepl
i
cat
eand
des
troyt
hec
ell
.

Gener
aldr
awbac
ksofv
iralv
ect
ors
:

 Av
irusc
an'
t"ex
pand"t
ofitapi
eceofgenet
i
cmat
eri
all
argert
hani
ti
snat
ural
l
ybui
l
t
t
ocar
ry.Ther
efor
e,s
omegenesmaybet
oobi
gtofi
tint
oac
ert
aint
ypeofv
irus
.

 Vi
rus
escanc
aus
eimmuner
espons
esi
npat
i
ent
s,r
esul
t
ingi
ntwopot
ent
i
al
out
comes:

1'
 Pat
i
ent
sma
ygets
ick.

 Apat
i
ent
'
simmuni
t
ytoav
irusma
ypr
eventhi
mfr
om r
espondi
ngt
orepeat
ed
t
reat
ment
s.

However
,moder
nvir
alvec
tor
sha vebeenengi
neer
edwi
t
houtmos
toft
hepr
otei
ns
t
hatwoul
dcauseanimmuneresponse.

(on)&iral &ectors

Al
t
houghv
irus
esc
aneffec
tiv
elydel
i
vergenet
i
cmat
eri
ali
ntoapat
ient
'
scel
l
s,t
heydo
ha
ves
omel
i
mit
ati
ons
.Iti
ssome
timesmor
eeffic
ientt
odel
i
verageneus
inganon-
v
iral
vec
tor
,whi
chhasf
ewers
izec
ons
trai
ntsandwhi
chwon'
tgener
ateani
mmune
r
esponse.

Non-vi
ralv
ectorsar
etypi
call
ycir
cularDNAmol ec
ules
,alsok
nownaspl
asmi
ds.I
n
nat
ure,bact
eriausepl
asmidstotransf
ergenesfr
om cel
lt
ocell
.

Scient
is
tsusebact
eri
aandplas
mi dst
oeas
il
yandeffic
ient
l
yst
oreandr
epl
i
cat
e
genesofint
eres
tfr
om an
yorganis
m.

Vec
tor
susedatpr
esent,ar
eengineer
edinsuchawaythatt
heyhelpeas
yli
nki
ngof
f
ore
ignDNAandselect
ionofr
ecomb i
nant
sfr
om non-
recombi
nant
s.

 These are not the only way to introduce alien *(A i

nto
ho
stc
ell
s.I
name
thodk
nowna cr
smi o-
inj
ect
ion,r
ecombi
nantDNAi
sdi
rec
tl
y
i
nject
edint
othenucl
eusofananimalcel
l
.Inanot
hermethod,s
uitabl
ef orplants
,
cel
lsarebombar
dedwit
hhighveloci
tymi
cro-
part
i
clesofgol
dortungstenc oatedwi
t
h
DNAi namethodknownasbiol
isti
csorgenegun.

Delivery to s&eci,c tiss(es

Del
i
ver
inggenest
os pec
ifict
is
sueswit
hinapat
ient
'sbodycanbever
ydifficul
t
.
Del
i
ver
inggenesi
ntoagroupofcel
l
sinapati
ent'
sbodycanbedoneinoneoftwo
way
s.

11
Thefirstwayist
oinjectt
hev ectori
ntot
hebodyandspeci
fical
lyt
argetaffec
tedcel
l
s.
Thisi
scall
edaninv i
vo appr
oac h.Thesec
ondway,c
all
ede xvi
vo,i
stodeli
vert
he
genetocell
swhil
ethey'r
eout s
idethebodyby:

 I
sol
ati
ngt
hedes
iredc
ell
sfr
om t
hebody
.

 Cul
t
uri
ngt
hecel
l
sinaPet
ridi
shi
nthel
abor
ator
y.

 Del
i
ver
ingt
hegenest
othec
ell
s(us
ingoneoft
hev
ect
oropt
i
onsdes
cri
bed
onthi
spage)
,ac
tiv
ati
ngt
hem,andmak
ings
uret
hatt
hec
ell
sint
egr
atet
hem
pr
operl
y.

Case +tudy ,
Cystic -ibrosis

%he Disease – ' #enetic Disor$er

Cy
sti
cfibr
osi
s(CF)
,al
sok
nownasmuc
ovi
sci
dos
is,i
sanaut
osomal
rec
ess
ive
genet
icdi
sor
dert
hataffec
tsmos
tcr
it
ic
all
ythel
ungs,andal
sot
hepancr
eas
,li
ver
,
andi
ntes
ti
ne.I
tischar
act
eri
zedbyabnor
malt
rans
por
tofc
hlor
ideands
odi
um ac
ros
s
anepi
t
hel
i
um,l
eadi
ngt
othi
ck,vi
scoussecr
eti
ons,pr
event
i
ngt
hec
il
iaf
rom cl
ear
ing
de
bri
swhi
chc
auses
ympt
omss
uchasc
oug
hin
g,p
oordi
ges
ti
onandi
ncr
ease
d
v
ulner
abi
l
it
ytoi
nfect
ion.

12
 CFi
scaus
edbyamut
ati
oni
nthegenef
ort
he
pr
otei
ncy
sti
cfibr
osi
str
ans
membr
anec
onduc
tanc
e
r
egul
ator(
CFTR)geneonchr
omosome7.Most
commonl
y,t
hemut
ati
oni
ntheCFTRgenei
sa
t
hree-
base-
pai
rdel
eti
on.Thi
spr
otei
nisr
equi
redt
o
r
egu
lat
eth
ecompo
nent
sofs
weat
,
di
ges
ti
vefl
uids
,andmuc
us.
CFTRr
egul
atest
he
mo
vementofc
hlor
ide
andsodi
umi
ons
acr
ossepi
t
hel
i
al
membr
anes,suc
has
t
heal
veol
arepi
t
hel
i
a
l
ocat
edi
nthel
ungs
.Si
nceal
loft
hec
ell
sofaCF
pat
ienthav
ethedef
ect
ivepr
otei
n,l
argequant
i
tiesof
t
hic
k,s
tic
kymuc
usbui
l
dupt
hroughoutt
hel
ungs
andot
heror
gans
.Thi
sres
ult
sint
hes
ever
it
yofs
ympt
omsseeni
nCFpat
i
ent
s.

Is It ' #oo$ %arget -or #ene

%hera&y.
Tocheckt
hissomequest
i
onsmustbeanswer
ed:

 *oes the condition result rom mutation.  

Yes.

 Is the biology o the disorder known.  

Yes.

 /ill adding a normal copy o the gene f0 the problem in the

aected tissue. 
Yes
.Wh
il
eth
emu
tat
edCFTRg
enee
nco
desan
on-
func
ti
ona
lio
n
c
hannel
prot
ein,i
twi
l
lnotpr
eventanor
mal
CFTRc
hannel
pro
tei
nfr
om wor
king
p
rope
rly
.Th
eref
ore
,add
ingan
orma
lcop
yoft
heCFTRg
enes
hou
ldfi
xthepr
obl
em

 Is it easible to delier the gene to the cells o the aected tissue.

Yes
,inpar
t.Tr
eat
i
ngt
hel
ungsofpat
i
ent
swi
t
hCFmi
ghtbef
eas
ibl
e,s
inc
ethel
ung

13
 s
urf
ace
sar
eex
pose
dtot
hea
ira
nds
ome
whatea
syt
ore
ach.Be
cau
set
hed
iges
ti
ve
s
yst
em i
sles
sac
ces
sibl
e,howev
er,i
tmi
ghtbeamor
edi
fficul
tregi
ont
otr
eat
.

Henc
ewec
anc
onc
ludet
hati
ti
saper
fec
tdi
seas
etobet
reat
edb
ygenet
her
apy
.

Choosing vectors
Thev
ect
orst
hatar
emos
tsui
t
abl
eforgenet
her
apyar
e:

Retrovir(s
Ret
rov
irus
esar
eenv
elopedv
irus
est
hatr
epl
i
cat
einahos
tcel
lt
hrought
hepr
oces
sof
r
ever
set
rans
cri
pti
on.I
tisas
ingl
e-s
trandedRNAv
irust
hats
tor
esi
t
snuc
lei
cac
idi
n
t
hef
orm ofanmRNAg
enomet
arge
ts.On
cei
nsi
det
heh
ostc
ellcy
topl
asmt
hev
irus
usesi
t
sownr
ever
set
ranscr
ipt
aseenzymet
opr
oduceDNAf
romi
t
sRNAgenome,
t
her
ever
seoft
heus
ualpat
ter
n,t
husr
etr
o(bac
kwar
ds)
.Thi
snewDNAi
sthen
i
ncor
por
atedi
ntot
hehos
tcel
lgenomebyani
ntegr
aseenz
yme,atwhi
chpoi
ntt
he
r
etr
ovi
ral
DNAi
sref
err
edt
oasapr
ovi
rus
.Thehos
tcel
lt
hent
reat
sthev
iralDNAas
par
tofi
tsowngenome,t
rans
lat
ingandt
rans
cri
bingt
hev
iralgenesal
ongwi
t
hthe
c
ell
'
sowngenes
,pr
oduc
ingt
hepr
otei
nsr
equi
redt
oas
sembl
enewc
opi
esoft
he
v
irus
.

Onedr
awbac
kofr
etr
ovi
rus
es,s
uchast
heMol
oneyr
etr
ovi
rus
,inv
olv
est
he
r
equi
rementf
orc
ell
stobeact
iv
elydi
vi
dingf
ort
rans
duc
tion.Asar
esul
t,cel
l
ssuchas
neur
onsar
ever
yres
ist
antt
oinf
ect
ionandt
rans
duc
tionbyr
etr
ovi
rus
es.

Butt
heai
rwa
ycel
l
swhi
char
eaff
ect
edb
ythedi
seas
ecy
sti
cfibr
osi
sandmus
tbe
t
arget
eddi
vi
dei
nfr
equent
l
y.Henc
eRet
rov
irusi
snotas
uit
abl
evect
orf
ort
hisdi
sease.

'$enovir(s
Adenovi
ruses(memb erso
fthefamilyAdenovi
ri
dae)ar
eme di
um-si
zed(90–
100nm)
,
nonenv
eloped(wit
houtanouterli
pidbil
ayer
)vi
ruseswit
hanic
osahedr
al
nucl
eocapsidcont
aini
ngado ubl
es t
randedDNAg enome.

1
Theyhav eabroadr angeofv ertebratehostsandha
vebee
n f
oundt
o
causeawi derangeofi l
lnesses,from mild
respi
rat
oryinf
ecti
onsi nyoungc hildrentoli
fe-
t
hreateni
ngmul t
i-
organdi seasei npeoplewi t
ha
weakenedi mmunes ystem.

Buttheycancause/
induceani
mmuneresponsei
nthe human
bodyhencenotsuit
ablef
orgenedel
i
ver
y.

/er&es Sim&le0 Vir(s

Her
pessi
mpl
exvi
ruses,al
soknownasHuman
her
pesv
irus
,ar
emember
soft
heher
pesv
irus
f
ami
l
y,Her
pes
vir
idae,t
hati
nfec
thumans
.They
canbespr
eadwhenani
nfect
edper
soni
s
pr
oduc
ingands
heddi
ngt
hev
irus
.Her
pes
Si
mpl
exc
anbes
preadt
hroughc
ont
actwi
t
h
s
ali
va,s
uchasshar
ingdr
ink
s.

Butt
hes
evi
rus
esonl
yaffec
tthec
ell
soft
hener
voussy
stem andc
annoti
nfec
tthe
ai
rwa
ycel
l
sandhenc
enots
uit
abl
e.

'$eno1'ssociate$ Vir(ses

Adeno-
associ
atedvi
rus(
AAV)
 i
sas
mal
lvi
rus
 whi
chi
nfect
shumansandsome
ot
herpr
imat
espec
ies
.AAVi
snotc
urr
ent
l
yknownt
ocaus
edi
seas
e and
c
ons
equent
l
ythev
irusc
aus
esav
erymi
l
dimmuner
espons
e.AAVc
ani
nfec
tbot
h
di
vi
dingandqui
escentc
ell
sandper
sis
tinanex
trac
hromos
omals
tat
ewi
t
hout
i
ntegr
ati
ngi
ntot
hegen
ome 
oft
hehos
tcel
l
.Des
pit
eit
sfewdi
sadv
ant
agest
hes
e
f
eat
uresmak
eAAVav
eryat
tr
act
iv
ecandi
dat
eforc
reat
i
ngv
iral
vec
tor
sfor
 gene
t
her
apy
,andf
ort
hecr
eat
i
onofi
sogeni
chumandi
seasemodel
s

ence it is the .est choice for ene dei/ery in the case of 5ystic 6i.rosis

1!
/istory o! Cystic -i2rosis #ene
%hera&y

Genether
apyforcysti
cfibr
os i
sbegani
n1990,whensci
enti
stssuccessful
ly
cor
rect
edfaul
tycy
sticfibr
osistr
ansmembr
aneconduc
tanceregulat
or(CFTR)genes
.
Theydidt
hisbyaddingnormalcopi
esoft
hegenetol
aborat
orycellcul
tures.

3445
I
n1993,t
hefirstex
peri
mental
CFgenet
her
apyt
reat
mentwasgivent
oapati
entwi
t
h
c
yst
icfibr
osi
s.Resear
cher
smodiedacommoncol
fi d Adeno
vir
ustoactasa
deli
ver
yv ehicl
ebyc arr
yingnormalgenestotheCFTRc ell
sinthenasal passages.
Researcherschosenas alpassagesasthesit
eofdel
iv
erybec ausethe
yar eeasiert
o
accessandmeas uregeneacti
v i
tyt
hanthelungai
rway.Latert
ri
alsdel
iveredthe
vect
ortopatients
’lungairway
s.

I
nt heearl
iertr
ials,i
thadl ookedlikethev i
rushadenteredc el
lsandthattheCTFR
genewaswor ki
ng.Butl atertr
ial
swi t
hdi ff
erentpati
entsshowedl evel
sofVFTRgene
act
ivi
tythatweret oolowt omak eanydi fference.Researcherscamet othi
nkthatt
he
adenovir
usc an’teasil
yent erair
wayc ell
s,especial
l
yinthelowdos esthatwerebeing
gi
ven.Intheear li
ertri
als,theyspeculat
ed,geneac ti
vi
tyresult
edf r
om thedamaget o
t
hec ell
sduringdel i
veryallowingthev i
rustoentereasi
ly.

Hencewhenhigherdosesofthevir
uswer
etri
ed,t
hei
mmunesy st
em oft
hepat
i
ent
s
st
art
edmounti
ngimmuner espon
sesandfight
i
ngoffthevi
rus.Thi
scause
da
bl
ockagei
nthetri
alsunti
l1998.

3446
T
rial
sus
ingAdeno-
ass
oci
atedv
irust
odel
i
vert
heCTFRgenebegani
n1998.Unl
i
ket
he
a
deno
vir
us,t
heAd
eno-
ass
oci
ate
dvi
rusc
aus
edn
oimmu
ner
espo
nseo
radv
ers
esi
dee
ffec
ts
i
npat
i
ent
s.

Butunl
i
ket
her
esear
cher
s’pr
edi
ct
ions
,theadeno-
assoc
iat
edv
irusdi
dnotent
erc
ell
s
effic
ient
l
yandi
ntegr
atei
ntocal
l
s’genomi
cDNA.The
ypr
oduc
edonl
ylo
w andflee
ting
amount
sofCFTRgeneac
ti
vi
ty
.Res
ear
cher
sar
est
i
llwor
kingt
ofigur
eoutwhatc
aus
edt
he
v
irus
est
ofai
l
.

1"
Butbec
aus
eiti
ssaf
e,t
hev
irus–aswepr
edi
ct
edear
li
er–hol
dspr
omi
sef
orbei
ngagood
wa
ytodel
i
vert
heCFTRgenet
opat
i
ent
s’ai
rwa
ycel
l
s.Butr
esear
cher
sneedt
olear
nmor
e
abouthowt
hev
irusi
nfec
tscel
l
sinor
derormak
eitaneffec
ti
vedel
i
ver
yme
thod.

Challenges
Somethefact
orst
hathav
eke
ptg
enet
her
apyf
rom b
ecomi
nganeff
ect
ivet
reat
ment
f
orgenet
icdis
easesar
e:

• +hort)lied nature o gene therapy ) Bef

oregenet
her
apycan
becomeap er
man entc ur
eforan ycondit
ion,thetherapeut
icDNAi nt
roduced
i
ntotar
getcel
l
smus tremai nfuncti
onalandt hecellscont
ainingthe
t
herapeut
icDNAmus tbelong-li
vedands table.Problemswi t
hintegr
ati
ng
t
herapeut
icDNAi ntot hegenomeandt her api
dlydivi
dingnatureofman yc el
ls
pr
eventgenetherapyf rom ac
hievinganylong- t
erm benefit
s .Pati
entswillhav
e
t
oundergomul t
ipl
er oundsofgenet her
apy .

• Immune response )Anyt

imeaf
orei
gnobj
ecti
sint
roduc
edi
ntohuman
t
issues,t
heimmunes yst
em isdesi
gnedt oatt
ackthei
nvader.Theriskof
st
imulati
ngtheimmu nes y
stem i
nawa ythatreducesgenetherapy
effecti
venessisalwaysapotenti
alr
isk
.Fur t
hermore,t
heimmunes ystem's
enhancedresponset oi
nvadersi
thass eenbeforemakesitdifficul
tforgene
t
herapytober epeatedinpati
ent
s.

• roblems with iral ectors ) Vi

ruses,whi
l
ethecar
ri
erofchoi
cei
n
mos tgenet herapystudi
es,presentavari
etyofpot enti
alprobl
emstot
he
pat
ient- -
toxi
city
,immuneandi nflammator
yr espons es,andgenecont
roland
t
argetingissues .I
naddit
ion,ther
eisalwayst hefearthatthevi
ralv
ect
or,onc
e
i
nsidet hepatient,mayrecoverit
sabil
i
tytoc ausedi sease.

• 4ultigene disorders )Condi

t
ionsordi
sor
der
sthatar
isef
rom mut
ati
ons
i
nas inglegenearethebes tcandi
datesforgenetherapy
.Unfort
unately,s
ome
t
hemos tcommonlyoccurr
ingdisorders,suc
hashear tdi
sease,highblood
pres
sure,Alzhei
mer'
sdiseas e,art
hri
ti
s,anddiabet
es,arecausedbyt he
combinedeffec t
sofvari
ati
onsi nmanygenes .Multi
geneormulti
fact
orial

1#
 di
sor
derssuchast
hes
ewoul
dbees
pec
ial
l
ydi
fficul
ttot
reateffec
tiv
elyus
ing
ge
netherapy
.

Issues regarding
Gene Therapy
Whatarethepossibl
eimpl
ic
ationsofgenet
her
apyresear
chtosoci
ety?Allofus-
res
earcher
s,pol
icymaker
sandt hepubli
c-h
avearesponsi
bil
i
tyt
oex pl
orethe
pot
enti
aleffect
sofgenetherapyresear
chonourl
i
vess ot
hatwecanmak einf
ormed
deci
si
ons.

Fore
achne
w ap
pli
cat
i
onofg
enet
her
apyr
esea
rch,wemus
tco
nsi
der
:

 Whatar
ethebenefit
s?

 Whatar
ether
isk
s?

 Whom wi
l
lthet
echnol
ogyhel
p?Whowi
l
litpot
ent
i
all
yhur
t?

 Whatdoesgenet
her
apymeanf
orus?

Ther
ear
esev
eral
typesofi
ssuest
ocons
ideraswet
hinkaboutgenet
her
apy
:

• Et
hicali
ssuesaskust
ocons
idert
hepot
ent
i
almor
alout
comesofgene
t
herapyresear
ch.

• Legalis
suesr
equi
reres
earcher
sandt hepubli
ctohel
ppoli
cymak
ersdec
ide
whetherandhowgenether
apyresear
c hshoul
dberegul
atedbyt
he
gover
nment.

• Soci
ali
ssuesi
nvol
vet
hei
mpac
tofgenet
her
apyr
esear
chons
oci
etyasa
whol
e.

1$
 Somequest
ionst
oponder

 /hen should gene therapy be used. Shouldi

tbeus
edt
otr
eatc
rit
i
cal
l
yil
l
pat
ient
s?Shoul
ditbeus edtotreatbabiesandchil
dren?

 /hat eect would gene therapy hae on uture generations i

germline 5reproductie6 cells were genetically altered. 
How mi
ghtt
his
al
ter
ati
onaff ecthumanv ariat
ion?

 /ho should decide what are 7good7 or 7bad7 uses o genetic

modifcations. 
Howd oy oudefine" normal
"wi t
hr egardtohu ma nbeings ?

 /hat i we could alter human traits not associated with

disease. 
Wo ulditbeoka ytou segenethe rapytoi mproveore nhanceape
rson
's
geneti
cprofile?

 /ho will hae access to gene therapy8 treatments and long)term

ollow)ups. 
Wi llgenether
apyandg eneti
cenhanc ement
screateana dvantagef
or
thosewhoc ana ffordi
t?

9ecent :pcoming
CRISPR

1%
CRI
SPRs
tandsf
orc
lus
ter
edr
egul
arl
y
i
nter
spac
edshor
tpal
i
ndr
omi
crepeat
s.
TheseRNAs
equenc
esse
rveani
mmune
f
unc
tioni
nar
chaeaandbac
ter
ia,buti
n
t
hel
asty
earors
o,s
cient
i
stshav
esei
zed
upont
hem t
orewr
it
egenes.TheRNA
s
equ
enc
eser
vesa
sag
uidet
ota
rge
ta
DNAsequencei
n,say
,azygot
eora
s
tem c
ell
.Th
egu
ideseq
uenc
elea
dsan
en
zyme,Ca
s9,t
oth
eDNAo
fin
ter
est
.
Cas
9canc
utt
hedoubl
est
rand,ni
cki
t
,or
e
venk
noc
kdo
wng
enee
xpr
ess
ion
.Af
terCas
9in
jur
est
heDNA,r
epa
irs
yst
emsfi
x
t
hesequence-ornewsequencescanbei
nser
ted.

I
tisn'tt
hefi r
storonlymethodofgener epairt
herapythat’
sbeendev el
oped,butthe
CRISPRt echnol
ogy,saysRa mes ar
,issospec i
albecause,unli
kepr ev
iousme t
hods
whichwer emorelabor
iousandc ouldonlytar
getonek i
ndofc elli
nthebody ,i
t
appearstobea" onesizefitsalldeli
ver
y",adaptabl
efordifferentt
issues.The
procedurealsoseemsrelat
ivel
ys i
mpletoperform.

Exci
ti
ngast
hede
vel
opmentma
ybe,CRI
SPRwon
’tbedel
i
ver
ingi
nst
antc
uresj
ust
ye
t.

Ramesarsays,f
rom hi
sini
t
iali
mpres
sionsoft
heli
t
erat
ure,t
hatitwoul
ds eem t
hat
l
ocal
is
ed,accessibl
eabnor
mal t
is
sue(asint
heret
inaorski
n)couldbetarget
ed
moreeasi
l
y .

Condit
i
onsaff ect
ingt
hebodymor es yst
emicall
y,howev er,s
uchasc er
tai
n
dev
elopmentalsyndr
omes,orcentr
al nerv
ouss ystem dis
order
s,mightbe
pr
oblemati
cintermsofget
ti
ngther epairt
echnologyintoenoughofthetar
getc
ell
sin
t
hatti
ssuetomak eaneffect
ivedifference.

"
Itmayals
odependonthestageoneat
temptstocarr
youtthether
apy
,inter
msof
t
hepati
ent’
sageandl
evelofadvanc
ementofthedis
ease,
"s ay
sRames ar
.

2'
Conclusion
Al
t
houghear
lycl
i
nic
alf
ail
uresl
edmanyt
odi
smi
ssgenet
her
apyasov
er-
hyped,
c
li
nic
als
ucc
ess
ess
inc
e2006ha
vebol
st
erednewopt
imi
smi
nthepr
omi
seofgene
t
her
apy
.Thes
einc
ludes
ucces
sful
treat
mentofpat
ient
swi
t
hther
eti
nal
di
seas
eLeber
'sc
ongeni
t
alamaur
osi
s,X-
li
nkedSCI
D,ADA-
SCI
D,
adr
enol
euk
ody
str
oph
y,c
hroni
cly
mphoc
yti
cleuk
aemi
a (
CLL)
,ac
utel
ymphoc
yti
c
l
euk
aemi
a (
ALL)
,mu
lti
plemy
elo
ma,haemophi
l
ia 
andPar
kins
on'
sdi
seas
e.These
r
ecentc
li
nic
als
ucces
seshav
eledt
oar
enewedi
nter
esti
ngenet
her
apy
,wi
t
hsev
eral
ar
ti
cl
esi
nsci
ent
i
ficandpopul
arpubl
i
cat
i
onscal
l
ingf
orcont
inuedi
nvest
menti
nthe
fi
eld.

;ibliography

7e2sites
• http<==en>wikipedia>org=wiki=Gene?therapy
• http<==www>trip2medi>com=treatmentCGeneTherapy>php
• http<==learn>genetics>utah>edu=content=tech=genetherapy=
• http<==ghr>nlm>nih>go=handbook=therapy=
• http<==cystic)fbrosis>emedt>com=cystic)fbrosis=cystic)fbrosis)gene)
therapy>html
• http<==en>wikipedia>org

Books
12th (C@9T ;iology
+tryer ;iochemistry

21