E D U C A T I O N A L SERIES R e d series"

Viral gene therapy

Pablo M a n c h e f i o - C o r v o and Pilar M a r t i n - D u q u e

l)pto, de Biotecnologia. Universidad Francisco tie Vitoria. Pozuelo de Alarc6n, Madrid. Spain.

~iding and d i v i d i n g cell types, but h a s a l i m i t e d

C a n c e r is a m u l t i g e n i c d i s o r d e r i n v o h ' i n g m u t a -
DNA capacity.
lions o f both t u m o r s u p p r e s s o r g e n e s a n d o n c o -
This r e v i e w discusses c u r r e n t a n d e m e r g i n g v i r u s -
genes. A l a r g e body o f p r e c l i n i c a l data, h o w e v e r ;
based g e n e t i c e n g i n e e r i n g strategies f o r the delivet w
has s u g g e s t e d that c a n c e r g r o w t h can be a r r e s t e d o r
of t h e r a p e u t i c m o l e c u l e s o r several a p p r o a c h e s for
r e v e r s e d by t r e a t m e n t with g e n e t r a n s f e r vectors
c a n c e r t r e a t m e n I.
that carry a s i n g l e g r o w t h i n h i b i t o r y o r p r o - a p o p -
totic g e n e o r a g e n e t h a t can r e c n t i t i m m u n e r e -
Key w o r d s : g e n e therapy, virus, a d e n o v i r u s , cancer.
sponses a g a i n s t the t u m o r :
Many of these gene transfer vectors ate modified Manche~o-Corvo P, Martin-Duque P I ))~d gene therapy. Clin
viruses. T h e ability for the d e l i v e t ) ~ o f t h e r a p e u t i c "D'ansl OncoL 2006;8(12):85 8~Z
genes, m a d e t h e m d e s i r a b l e for e n g i n e e r i n g v i r u s
v e c t o r systems. T h e viral vectors r e c e n t l y in l a b o r a -
tory a n d clinical use are based on RNA and DNA
viruses p r o c e s s i n g very different g e n o m i c s t r u c t u r e s
a n d host ranges. P a r t i c u l a r v i r u s e s h a v e b e e n se- INTRODUCTION
lected as g e n e d e l i v e o ~ v e h i c l e s b e c a u s e o f t h e i r ca-
Most c o n v e n t i o n a l drugs are proteins or small mole-
pacities to carry foreign g e n e s a n d t h e i r ability to
cules that interact with them. Thus, they act at the
efficiently d e l i v e r these g e n e s a s s o c i a t e d with effi-
protein level rather than at the u n d e r l y i n g level of
cient g e n e e x p r e s s i o n . T h e s e a r e the m a j o r r e a s o n s
genes. An alternative a p p r o a c h is gene therapy, de-
w h y viral v e c t o r s d e r i v e d f r o m retroviruses, a d e n -
fined as the use of nucleic acids to repair the malfunc-
ovirus, a d e n o - a s s o c i a t e d virus, h e r p e s v i r u s a n d
poxvilxts are e m p l o y e d in m o r e t h a n 70% o f clinical tioning DNA s e q u e n c e or to introduce a c o m p e n s a t o r y
c h a n g e that will restore the n o r m a l physiological
g e n e t h e r a p y trials w o r l d w i d e . B e c a u s e these v e c t o r
funclions of the cell. Gene therapy is one c o m p o n e n t
s y s t e m s h a v e u n i q u e a d v a n t a g e s a n d limitations,
of an e m e r g i n g group of therapies collectively de-
e a c h has a p p l i c a t i o n s For w h i c h it is best suited.
scribed as gene medicine. One form of gene m e d i c i n e
Retroviral v e c t o r s c a n p e r m a n e n t l y i n t e g r a t e into
the g e n o m e o f the i n f e c t e d cell, but r e q u i r e m i t o t i c is the use of nucleic acids in the s a m e m a n n e r as con-
cell division for t r a n s d u c t i o n . A d e n o v i r a l vectors ventional drugs, though the target of the therapy is
can efficiently d e l i v e r g e n e s to a w i d e variety o f di- the mRNA produced by the altered gene rather than
v i d i n g a n d n o n d i v i d i n g cell types, but i m m u n e the protein. Also the use of DNA vaccines, w h i c h ex-
e l i m i n a t i o n o f infected cells often l i m i t s g e n e ex- press antigens in the body, c o m e s u n d e r the h e a d i n g
p r e s s i o n in v i v o . H e r p e s s i m p l e x virus can d e l i v e r of gene medicine. All these new techniques are espe-
large a m o u n t s o f e x o g e n o u s DNA; h o w e v e ~ cyto- cially interesting for the t r e a t m e n t of diseases such as
toxicity a n d m a i n t e n a n c e o f t r a n s g e n e e x p r e s s i o n cancer, in w h i c h c o n v e n t i o n a l drug t r e a t m e n t is lira-
r e m a i n as obstacles. AAV also infects m a n y n o n - d i - fled
Gene transfer technology is b e c o m i n g i n c r e a s i n g l y
integrated with cell therapy; a therapeutic modality
based on the use of w h o l e cells derived either from
*Supported by an unrestricted educational ~ ' a n t from the patient or fi'om an alternative source. Both gene
AstraZeneca.
and cell therapy p e r h a p s r e p r e s e n t the most promis-
C o r r e s p o n d e n c e : P. Marlin-Duque. ing o f therapeutic strategies for long-term illnesses
Dpto. B i o t e c n . l o g i a .
U n i v e r s i d a d Francisco de Vitoria. such as cancer.
Ctra. M-515. Pozuelo-l~lajadah.nda, kill. 1,800. Viruses have been the most c o m m o n l y used vectors
28225 Pozuehl de A larc6n, Madrid. Spain.
E-mail: p.nlartin@ u fv.es for gene therapy because of their high efficiency of

~ Clin Transl Oitcol. 2006;8( 12): 858-67

 MANCHEI~O-CORVO P, MARTiN-DUQUE P. VIRAL GENE THERAPY
transduction into h u m a n cells, and about 70% o f g e n e
therapy clinical trials use viral vectors. We will re-
view some of those vectors in this article.
GENE TH E R A P Y
Gene therapy is a therapeutic strategy in which a pa-
tient's cells are genetically modified in an attempt to
cure a disease. There is an important distinction be-
tween somatic gene therapy, where modifications are
introduced into somatic cells a n d are confined to the
patient, and germ-line gene therapy w h e r e modifica-
tions are introduced into the cells that ~ v e rise to ga-
metes, and can therefore be passed to subsequent gen-
erations. Only somatic gene therapy is currently
permitted. While germ lille gene therapy has been
universally b a n n e d because of its ethical i replications.
Different types of somatic gene therapy approaches
for c a n c e r can be envisaged; their suitability depends
on the cancer's type.
Gene replacement therapy" tile aim is to introduce a
n o r m a l f u n c t i o n i n g copy of tile nonflmctional target
gene and then achieve the exogenous DNA to u n d e r -
go r e c o m b i n a t i o n with the target and therefore re-
place it. Although this is the most straightforward
way to correct genetic defects, the honlologous re-
c o m b i n a t i o n process is very inefficient in most cells.
Killing of specific cells: consists on e l i m i n a t i n g certain
population of cells, thus, it is very suitable for c a n c e r
gene therapy. The aim is to express within such cells
a suicide gene whose product is toxic, or to direct the
replication of viral vectors. O w i n g to the lethal effects
of suicide genes, they m u s t be directed to target cell
types with great accuracy to avoid side effects.
GENE DELIVERY MECHANISMS
The overall strategy for gene delivery comprises the
m e c h a n i s n l by which nucleic acids gain entry to the
cell. Gene transfer m e c h a n i s m s used for" gene therapy
are either viral or non-viral. Viral delivery, also
k n o w n as transduction, involves tile packaging of
DNA (or in some cases RNA) into a virus particle.
Gene transfer occurs by the n o r m a l viral infection
route and is both efficient and cell selective. For this
reason, viral delivel-y is the preferred strategy for in
vivo gene therapy.
Viruses are pathogenic entities; therefore, steps must
be taken to p r e v e n t the viral vector to cause disease.
Tile u s u a l method is to remove genes, which are es-
sential for virus replication; this approach also in-
creases the capacity of h a r b o u r i n g foreign DNA. The
m i s s i n g functions must be supplied fl'om all alterna-
tive source. This may m e a n u s i n g a helper virus to
make the m i s s i n g gene products but in most cases a
packaging line is used, i.e. a cell line that is stably
transformed with the appropriated viral genes. Once
Clin Transl Oncol. 2006;8(12):858-67
tile DNA is packaged, the defective virus can be iso-
lated from the cell lille. It carl then infect its target cell
and introduce its cargo of DNA or RNA, but it c a n n o t
replicate and cause disease symptoms.
G e n e t r a n s f e r eX-t'ilro or in-t,il,o
E x viro gene therapy involves transfer of cloned
genes into cells grown in culture. Those cells that
have been transformed successfully are selected, ex-
panded by cell culture in vitro and then replaced in
the patient. To avoid rejection by tile i m m u n e system,
the patient's o w n cells (autologous cells) are used
w h e n e v e r possible. This approach is used for cells
that are accessible for" initial r e m o v a l and that carl be
induced to engraft and survive for a long time after"
replacement. Examples include cells of tile hemato-
poietic system, skill cells, etc.
ht vivo gene transfer is the only option in tissues
where tile recipient cells c a n n o t be cultured in vitro
in sufficient n u m b e r s (e.g. brain cells) or where cul-
tured cells cannot be re-implanted efficiently in pa-
tients. Tissue targeting is an i m p o r t a n t consideration.
The gene transfer construct may be emplaced directly
into the target tissue, or it m a y be injected into the
general circulation but designed in some way so as to
be taken up only by the desired cell type (fig. 1).
Vector integration
For achieving long-term expression it would seem
desirable to integrate tile foreign gene into a c h r o n i c -
some of the host cells, preferably a stem cell. Then,
the construct is replicated w h e n e v e r the host cell or
its daughters divide (fig. 2).
However, integration carries certain p r o b l e m s and
risks. Integration of most constructs occurs at r a n d o m
sites, and will be different in various cells of the pa-
tient. It may n e v e r be expressed, be expressed at un-
desirable low level, or may be expressed for" a short
time a n d then irreversibly silenced. Worse, the inte-
gration may alter expression of e n d o g e n o u s genes.
The greatest worry is that insertion of a highly ex-
pressed construct may activate an adjacent oncogene,
similar to the activation of Myc in Burkitt's lym-
phoma and indeed, this is precisely what seems to
have happened in two of the children successfully
treated for" severe c o m b i n e d i m m u n o d e f i c i e n c y12, .
Apparently, in at least one of the 10 ~ modified T cells
in each of the two children, a r a n d o m retroviral ill-
sertion had activated the LMO2 oncogene. Eventually
this clone outgrew all others, leading to a novel form
ofT-cell leukemia. It is likely that this experience will
lead to a general rejection of r a n d o m l y integrating
vectors as tools for gene therapy.
For all these reasons, vectors that r e m a i n as extra-
clu'omosomal episomes seem likely to become the
859
 MANCHEI~O-CORVO P, MARTiN-DUQUE P. VIRAL GENE THERAPY

Cioncd gcne
X
J ' - ~ ~ - Gene Select X +
~ transfer cells

Cells r e m o v e d ~ " s Some cells

~ ~ X- nowX+
Amplify

cells to patient X+cells

Fig. 1. In vivo and ex vivo gene therapy: where possible, cells are removed from the patient, modified in the laboratory
and returned to the patient (ex vivo gene therapy). This allows just the appropriate cells to be treated, and cells can be
checked before they are replaced to make sure that the desire change has been achieved. For many tissues this is not
possible and the cells must be modified within the patient's body (in vivo gene therapy).

m a i n s t r e a m gene therapy tools. Their d i s a d v a n t a g e is viruses: it i m p l i e s the r e p l a c e m e n t of the viral g e n t -

the limited d u r a t i o n of gene expression. If the target
cells are actively dividing, the e p i s o m e s will tend to
be diluted out as the cell population grows. T h u s
there is no possibility o f a c h i e v i n g a p e r m a n e n t cure,
and repeated t r e a t m e n t s m a y be necessary.
VIRUSES AS GENE THERAPY VECTORS
There is no an ideal gene transfer system, each has
its own limitations. However, m a m m a l i a n v i r u s e s
have b e e n tile most c o m m o n l y used vectors for gene
transfer b e c a u s e of their high efficiency of t r a n s d u c -
tion into h u m a n cells. About 70% of a p p r o v e d proto-
cols use viral vectors, h a v i n g developed a c o n s i d e r -
able n u m b e r of different viral s y s t e m s (fig. 3).
Oncoretroviral vectors
Retroviruses have been used as gene vectors t h r o u g h
the past decades. They are RNA viruses, with a sin-
gle-chain 10 kb RNA g e n o m e , w h i c h is converted to
DNA by m e a n s of a reverse t r a n s e r i p t a s e e n c o d e d by
the own virus. The obtained viral-DNA is able to i.nte-
grate in the host's cell genome.
Given the ability of native r e t r o v i r u s e s to transform
cells, they m u s t be m a n i p u l a t e d to m a k e their replica-
tion system defective. This can be achieved by replac-
ing the viral genes (the gag-pol-env genes) with those
that are to be i n t r o d u c e d (fig. 4). The construction of
a t r a n s m i s s i b l e retroviral vector that e x p r e s s e s the
t h e r a p e u t i c gene is quite s i m i l a r to the case of o t h e r
me with a p l a s m i d c o n t a i n i n g the transgene but con-
s e r v i n g the viral packaging, p r o m o t i n g and p o l y a d e -
nylation sequences. A c o o p e r a t o r or p a c k a g e r virus
p r o d u c e s the capsid p r o t e i n s that would form e m p t y
viral particles unless they c o m b i n e with the g e n o m e
constructed with the t h e r a p e u t i c gene, which is de-
fective in t e r m s of replication 5. This e n s u r e s the first
wave of infection and the incorporation in the trans-
ferred cells g e n o m e with no risk of s e c o n d a r y re-in-
fections. The p r e s e n c e of viral p r o m o t e r s e q u e n c e s
and a n y added s e q u e n c e s of m a m m a l i a n or h u m a n
origin, together with the therapeutic gene, can confer
substantial cell line specificity to the gene 4.
Integration requires the r e t r o v i r a l eDNA to gain ac-
cess to the host c h r o m o s o m e s , w h i c h is possible only
when the n u c l e a r m e m b r a n e dissolves d u r i n g cell di-
vision; thus, r e t r o v i r u s e s can only infect dividing
cells. The p r o p e r t y of t r a n s d u c i n g only dividing cells
can be turned into an a d v a n t a g e in c a n c e r treatment.
Actively dividing c a n c e r cells in a nornlally non-di-
viding tissue like brain can be selectively infected and
killed without m a j o r risk to the nornlal cells.
The use of retroviral vectors to transfer suicide genes
in selective ehemotlaerapy is a d v a n t a g e o u s b e c a u s e of
that ability to infect only those cells that are u n d e r g o -
ing proliferation. This is evident in the case o f the
glioma 5, which has been tile subject of clinical trials
with t h y m i d i n e kinase of HSV, involving the intra-
c e r e b r a l i m p l a n t a t i o n of p r o d u c e r and p a c k a g e r cells
and the administration of gancielovir. Clinical gene
therapy trials u s i n g r e t r o v i r u s e s are c u r r e n t l y ongo-

860 Clin Transl Oncol. 2006,8(12):858-67

 MANCHEIqO-CORVO P, MARTiN-DUQUE P. VIRAL GENE THERAPY

Cloned Gene

" . P o r o u s nuclear
ear elope

I I
Integrated Episomal
gene gene

if

:i
':i f'

/ ",,. Cell Division /

>

Fig. 2. Integration or Episomal transmision of the foreign gene into the daughter cells: for achieving long term expres-
sion it would seem desirable to integrate the foreign gone into a chromosome of the host cells, otherwise the expres-
sion would be lost whenever the host cell or its daughters divide as figure shown.

ing for t r e a t m e n t of d i s e a s e s r e q u i r i n g the inhibition
of gene e x p r e s s i o n and as a genetic i m m u n o m o d u l a -
tion t h e r a p y to replace lost s u p p r e s s o r genes 6.
Lentiviral vectors
These are specialized r e t r o v i r u s e s that have the use-
ful attribute of infecting n o n - d i v i d i n g cells 7. Like oth-
er retroviruses, they integrate into the host c h r o m o -
s o m e s at r a n d o m places, giving the possibility of
long-term gene e x p r e s s i o n but with all the safety im-
plications that integration carries. H u m a n HIV is the
basis of most lentiviral vectors, which u n d e r s t a n d -
ably p r o v o k e s n e r v o u s n e s s about the risk of i n a d v e r -
tently g e n e r a t i n g replication c o m p e t e n t virus. The
H1V g e n o m e is more c o m p l e x than s t a n d a r d retro-
viruses, and m u c h w o r k has been devoted to elimi-
nating u n n e c e s s a r y genes and g e n e r a t i n g safe pack-
aging lines w h i l e r e t a i n i n g the ability to infect
n o n - d i v i d i n g cells. Self-inactivating vectors p r o v i d e
an additional layer" of safety s.
Adeno~iral vectors
Adenoviruses are DNA viruses that cause benign in-
fections of the u p p e r respiratory tract in h u m a n s .
They can be p r o d u c e d m high titers and t r a n s d u c e ef-
ficiently both dividing and n o n - d i v i d i n g cells. Tile
linear 56 kb d o u b l e - s t r a n d e d DNA g e n o m e , r e m a i n s
as an e p i s o m e within the cell nucleus. T h e virion is a
700 n m - d i a m e t e r i c o s a h e d r o n exclusively m a d e up of
protein and DNA. Adenoviruses have b e e n isolated in
m a n y species, 4,3 in h u m a n and more than 100 differ-
ent serotypes have been identified; the most thor-
oughly c h a r a c t e r i z e d a d e n o v i r u s e s are serotypes 2, 5
and 12.
Adenoviruses a r e attracting attention as potential vec-
tors with e x p r e s s i o n in p r i m a r y cells and as r e c o m b i -
nant vaccines ff)r m a n y reasons. First, the viral parti-
cles a r e very stable and the foreign genes inserted are
m a i n t a i n e d t h r o u g h o u t successive r o u n d s of viral
replication, at least in the most c o m m o n l y used
serotypes 9. Moreover, the a d e n o v i r u s g e n o m e is rela-
tively easy to m a n i p u l a t e u s i n g r e c o m b i n a n t DNA
techniques, and the v i r u s replicates efficiently in per-
missive cells.
The replication cycle o f the v i r u s can be divided into
two phases: early, c o r r e s p o n d i n g to events o c c u r r i n g
p r i o r to the replication o f the viral DNA, and late, cor-
r e s p o n d i n g to the period following the start of viral
DNA replication. D u r i n g the early phase, four regions

C/in Transl Om'ol. 2006;8(12):858-67 86"1

 MANCHEI~O-CORVO P, MARTIN-DUQUE P. VIRAL GENE THERAPY

.,....>

Vector

Target Cell

Fig. 3. Basis of Viral Gene Therapy: viral delivery involves the packaging of DNA (or in some cases RNA) into a virus
particle containing the therapeutic transgene, that is able to infect the target cell, in order to correct the malfunction on
the cell.

(early regions, E) are expressed: E1 (EIA and E1B),
E2, E5 and E4 I~ (fig. 5).
A m o n g the regions of the viral genome, there are
three that are capable of accepting DNA insertions or
substitutions For" the generation of a r e c o m b i n a n t
vh'us: El, E5 a n d a small region located between E4
and the end of the genome.
As with retroviral vectors, adenoviral vectors are dis-
abled and rely on a packaging cell to provide vital
function. The El region is not necessary for viral
replication in h u m a n cell line 293 (which is trans-
formed by Ad5 DNA a n d express the left end of tile
genome), although it would be for the r e m a i n d e r of
the cell lines. E5 is not required for" adenovivus repli-
cation in h u m a n cultured cells.
The m a x i m u m a m o u n t of DNA that can be packaged
in virions is limited to a n extra capacity of about 2 kb
of extra DNA. To incorporate larger DNA segments, it

Lipid bilayer
Envelope
protein
(env)

C a p s i d proteir
(gag)

Reverse
transcriptas~
(pol)

Fig. 4. Retrovirus schematic: retroviruses are RNA viruses, with a single-chain 10 kb

RNA genome which is converted to DNA by means of a reverse transcriptase encod-
ed by the own virus. The obtained viraI-DNA is able to integrate in the host's cell
genome.

862 Clin Transl Oncol. 2006;8(12):858-67

 MANCHEI~O-CORVO P, MARTiN-DUQUE P. VIRAL GENE THERAPY

0 10 20 30 40 50 60 70 80 90 ..........
100 --!

(100 mu = 35 935 bp)

ITR,d
ITR
--~

EIA EIB EIII EIV

lip

Late transcription
Fig. 5. The earlyadenoviralgenome: transcription from the viral genome is divided in early and late phases. Early, cor-
responding to events occurring prior to the replication of the viral DNA, and late, corresponding to the period following
the start of viral DNA replication. During the early phase, four regions (early regions E) are expressed: E1 (EIA and
E1 B), E2, E3 and E4 (ITR, InvertedTermlnal Repeats; ~, packaging signal).

would be necessary to c o m p e n s a t e by deleting the ap-
p r o p r i a t e a m o u n t s of viral DNA 11. Gutless vectors
have all viral genes deleted and can a c c o m m o d a t e up
to 35 kb of therapeutic DNA.
Genetic targeting is a n o t h e r of the strategies c o m -
monly developed for a d e n o v i r a l c a n c e r gene thera-
pyl2.
The big p r o b l e m with a d e n o v i r a l vectors is their im-
m u n o g e n i c i t y 13. Even though a live replication c o m -
petent a d e n o v i r u s vaccine has been safely a d m i n i s -
tered to million US a r m y recruits o v e r several
decades, u n w a n t e d i m m u n e reactions have been a
p r o b l e m in s o m e gene t h e r a p y trials. In S e p t e m b e r
1999, a patient died two clays after receiving 6 x l015
r e c o m b i n a n t a d e n o v i r u s particles by i n t r a - h e p a t i c in-
jection in a gene t h e r a p y phase I trial. Patients in oth-
er trials have suffered lesser but significant inflam-
m a t o r y responses. Moreover, b e c a u s e these vectors
are non-integrating, gene expression is short termed.
Repeated a d m i n i s t r a t i o n would be n e c e s s a r y for sus-
tained expression, w h i c h could only e x a c e r b a t e the
i m m u n e response.
Adeno-associated vectors
Adeno-associated virus (AAVs) are n o n p a t h o g e n i c
s i n g l e - s t r a n d e d viruses that rely on co-infection by an
adeno or h e r p e s h e l p e r virus to replicate 14. U n m o -
dified h u m a n AAV integrates into c h r o m o s o m a l DNA
at a specific site on 19qlS.5-qrer. This is a highly de-
sirable property, p r o v i d i n g the a d v a n t a g e of long-
term expression without the risk of insertional muta-
genesis. Unfortunately, the specificity of integration is
provided by the viral protein rep, w h o s e coding gene
is deleted ill tile construct used for gene transfer. As
with o t h e r system, the functions n e c e s s a r y for pro-
duction of viral p r o t e i n s ( i n c l u d i n g rep) are p r o v i d e d
by a p a c k a g i n g cell. In AAV-based vectors, as m u c h
as 96% of the AAV g e n o m e has been deleted; this pro-
vides a high degree of safety b e c a u s e the r e c o m b i n a n t
vectors contain almost no viral genes. H o w e v e r , the
AAV g e n o m e is very small, and even these highly delet-
ed vectors can only accommodate inserts up to 4.5 kb.
The a p p l i c a t i o n s of r e c o m b i n a n t p a r v o v i r u s vectors
to c a n c e r t h e r a p y include: (A) Genetic m a r k i n g of
h e m a t o p o i e t i c cells. They seem p r o m i s i n g for gene
transfer to h e m a t o p o i e t i c progenitors and are cur-
rently u n d e r g o i n g intensive evaluation for in ~,il'o ef-
ficacy 15. (B) C h e m o p r o t e c t i o n o f h e m a t o p o i e t i c cells.
Augmented b,IDRI (multi d r u g resistance) expression
a m o n g hematopoietic p r o g e n i t o r s and their p r o g e n y
may abrogate the m y e l o s u p p r e s s i v e effects of dose-
intensified c h e m o t h e r a p y and p e r m i t m o r e intensive
and effective r e g i m e n s 16. (C) Delivery o f t r a n s d o m i -
nant molecules: they could be the delivery vehicle for
antisense transcripts 17 or r y b o z y m e s 18. (D) Modula-
tion o f a n t i t u m o r i m m u n i t y : Antigenic d e t e r m i n a n t s
must he solely or selectively expressed on the t u m o r
as c o m p a r e d to n o r m a l cells fur an ant_itumor i m m u n e
response to be mounted 19
Togavirus vectors
The T o g a v i r i d a e family i n c l u d e s two types of RNA
viruses, the a l p h a v i r u s e s and the rubiviruses. The

Clht Transl Oncol. 2006;8(12):858-67 863

 MANCHEIqO-CORVO P, MARTIN-DUQUE P. VIRAL GENE THERAPY
two most widely studied a l p h a v i r u s e s are the Sin(Ibis
virus (SIN) and the Semliki Forest virus (SFV), w h i c h
have been used for heterologous gene e x p r e s s i o n -'2~
In nature, a l p h a v i r u s e s are t r a n s m i t e d by mosquitoes
to vertebrate hosts; in culture, they can infect cells
from a large variety of a n i m a l s , fl'om insects to m am-
reals. The infection o f cultured vertebrate cells is
c h a r a c t e r i z e d by a d r a m a t i c cytopathic effect and rap-
id cell death, while their growth in mosquito cells al-
lows t h e m to establish chronic or persistent infec-
tions 21.
The g e n o m e of tile a l p h a v i r u s e s consists of a single-
chain RNA with positive polarity. The g e n o m e is di-
vided in such a w a y that the replication proteins are
encoded by an o p e n - r e a d i n g f l a m e (ORF) in the ge-
nomic DNA, while the structural proteins are encoded
by a second, separate ORF. This permits the develop-
ment of s u b g e n o m i c s e q u e n c e s that call be m a n i p u -
lated with no i m p a c t on the replication capacity o f the
s y s te m 22.
Although s o m e a l p h a v i r u s e s are pathogenic, the two
that are used a s vectors, SFV and SIN, are n o n - v i r u -
lent in l m m a n s . For reasons of biosafety, and b e c a u s e
the large-scale production of RNA in vitro w o u l d be
costly, s o m e l a b o r a t o r i e s have developed a new strat-
egy with a DNA-RNA vector system. This system,
w h i c h is i n d e p e n d e n t of h e l p e r vectors, has tile cas-
sette of r e c o m b i n a n t a l p h a v i r u s cDNA expression
controlled by a e u k a r y o t i c promoter. This c o m p l e x is
i n t r o d u c e d into the cell by c o n v e n t i o n a l DNA trans-
Fection methods. In the cell nucleus, the
p o l y m e r a s e RNA t r a n s c r i b e s the complete unit into
RNA, w h i c h is t r a n s p o r t e d to the c y t o p l a s m 22. As a
result of its positive polarity, the RNA is converted to
viral replicase, which e l i m i n a t e s the replication o f the
molecule itselF, just like d u r i n g normal replication of
the a l p h a v i r u s RNA molecule.
Vaccinia v i r u s v e c t o r s
M e m b e r s of tile Poxviridae family of viruses, includ-
ing vaccinia, are u n u s u a l in the w a y that replication
and t r a n s c r i p t i o n of the g e n o m e occurs in the cytosol
of infected cells, with virally encoded p o l y m e r a s e s
d r i v i n g these processes. Thus, r e c o m b i n a t i o n of viral
DNA into tile g e n o m e is not a concern with vaccinia
vectors, as it is with others, p a r t i c u l a r l y retroviruses.
The p o x v i r u s family is s u b d i v i d e d ill two subfamilies:
E n t o m o p o x v i r i n a e (insect poxvirus) and Chordopo-
xvirinae (vertebrate poxvirus). Vertebrate poxviruses
share a group-specific antigen and are c a p a b l e of pro-
d u c i n g n o n g e n e t i c reactivation of other viruses be-
longing to the s a m e group 23.
Poxviruses are the largest-sized a n i m a l viruses, and
can be seen by light m i c r o s c o p y . The prototype of the
group, the vaccine virus, is 185 kb and tile virion con-
tains m o r e than 100 p o l y p e p t i d e s that are a r r a n g e d ill
864 Clin Transl Oncol. 2006;8(12):858-67
fbur different structures (nucleoids, lateral bodies,
m e m b r a n e and coating) 24.
The infectious cycle is divided into three phases.
During early phases, prior to replication, proteins
with e n z y m a t i c function are expressed. Tile expres-
sion of a small n u m b e r of i n t e r m e d i a t e genes de-
pends on replication of the genome; in turn, these in-
t e r m e d i a t e p r o d u c t s drives tile e x p r e s s i o n of a large
set of late genes, typically e n c o d i n g structural pro-
teins 25. G e n e r a l l y speaking, 4 a t e , vaccinia p r o m o t e r s
drive stronger gene e x p r e s s i o n than ~early, p r o m o t -
ers. Some genes, such as that e n c o d i n g the 7.5-kDa
protein, have both early and late promoters, and are
expressed d u r i n g both p h a s e s of infection, with late-
p r o m o t e r c o m p o n e n t a c c o u n t i n g for a p p r o x i m a t e l y
750/0 of total e x p r e s s i o n 26.
In recent years, n u m e r o u s genes that e n c o d e i m p o r -
tant proteins fi'om v i r u s e s and o t h e r m i c r o o r g a n i s m s
have been inserted into the vaccine v i r u s 27. The re-
sulting r e c o m b i n a n t s have been e m p l o y e d ill basic
studies d e a l i n g with gene expression, transcription,
protein processing, t r a n s p o r t and secretion of pro-
teins 28. Also they have been used ill i m m u n o l o g i c a l
studies, especially in the production of target cells ex-
p r e s s i n g surfime epitopes, which are useful for cellu-
lar i m m u n i t y reactions 29.
Vaccinia has b e e n extensively used in m a n ; 20 y e a r s
ago it was the keystone for the e r a d i c a t i o n of s m a l l -
pox (caused by the variola poxvirus) 5~ therefore, it is
well c h a r a c t e r i z e d in the field. A second i m p o r t a n t
characteristic is its stability: vaccinia call be carried
to r e m o t e regions and reconstituted fi'om dried mate-
rial to a highly infectious stock. Over time, vaccinia
has been recognized as a relatively safe agent with
infrequent s e r i o u s side effects, although it does in-
duce a vigorous i m m u n e response and call be lethal
[br those who are i m m u n o c o m p r o m i s e d or have ec-
zema 51.
Vaccinia is best k n o w n as a v e c t o r For transient ex-
p r e s s i o n of proteins. A clear a d v a n t a g e o f v a c c i n i a
in tiffs r e g a r d is its w i d e t r o p i s m : with v a r i a b l e effi-
ciency, v a c c i n i a infects most m a m m a l - d e r i v e d per-
m a n e n t cell lines. Its large g e n o m e a l l o w s the stable
insertion of very large f r a g m e n t s of DNA (25 kb has
been r e p o r t e d 32) into tile g e n o m e at a single site.
This is w e l l a b o v e tile range of m a n y o t h e r vectors.
Such r e c o m b i n a n t s h a v e been used for a wide r a n g e
of studies i n c l u d i n g those c o n c e r n i n g with Folding
and o l i g o m e r i z a t i o n o f p r o t e i n s 33, s i g n a l t r a n s d u c -
tion 54, s e v e r a l c y t o k i n e s 55 or by elicting protective
i m m u n e r e s p o n s e s a g a i n s t p a t h o g e n s and t r a n s -
f o r m e d cells 3~'37.
H e r p e s s i m p l e x ~irus ( h s v ) v e c t o r s
HSV vectors have t r o p i s m for the central n e r v o u s
system (CNS). Inset c a p a c i t y is at least 50 kb. T h e s e
 MANCHEI~O-CORVO P, MARTiN-DUQUE P. VIRAL GENE THERAPY
are complex v i r u s e s with a d o u b l e - s t r a n d e d DNA ge-
home of 152 kb c o n t a i n i n g at least 80 genes. They
can establish lifelong latent infections in sensory
ganglia in w h i c h they exist as n o n i n t e g r a t e d extra-
c h r o m o s o m a l elements. The latency m e c h a n i s m
might be exploitable to allow l o n g - t e r m expression
of transferred genes, hopefully s p r e a d i n g through a
synaptic network. Their m a j o r application w o u l d be
in d e l i v e r i n g genes into n e u r o n s for the t r e a t m e n t of
diseases such as P a r k i n s o n and CNS tumors. Prac-
tical vectors are still at an early stage of develop-
i n e n t 58.
ONCOLYTIC V E C T O R S FOR C A N C E R T H E R A P Y
Another growing area of gene therapy is the use of
oncolytic vectors for cancer treatnaent. Like im-
m u n o t h e r a p y , this is a concept that has been a r o u n d
for a h n o s t a c e n t u r y and it is u n d e r g o i n g a renais-
sance due to gene therapy 59.
Oncolytic gene therapy vectors are generally viruses
that have been genetically engineered to target and
destroy cancer cells while r e m a i n i n g i n n o c u o u s to
the rest of the body. Oncolytic vectors are designed to
infect c a n c e r cells and i n d u c e cell death through the
propagation of the virus, expression of cytotoxic pro-
teins a n d cell lysis 4~ A n u m b e r of different viruses
have been used for this purpose, i n c l u d i n g vaccinia,
adenovirus, herpes simplex virus type 1, reovirus and
Newcastle disease virus 41. These viruses have been
chosen, in m a n y cases, because of their nattu'al abili-
ty to target c a n c e r cells as well as for the easiness of
m a n i p u l a t i n g their genome.
M a m m a l i a n models of oncotytic gene therapy have
highlighted the potential of this approach. M u r i n e
models of colon and bladder c a n c e r have s h o w n sur-
vival benefits and reduced metastasis u s i n g oncolytic
viral agents 42,43. I11 a c a n i n e model, u s i n g an oncolyt-
ic virus, stuwival was prolonged even in imnauno-
competent dogs with syngenic osteosarcoma 44.
However, most people have antibodies to the com-
mon viruses used for therapy development, which of-
ten leads to an i m m u n e response that clears the viral
agent before it has had time to infect cancer cells 4"5. In
a trial u s i n g a modified vaccinia virus to treat breast
and prostate cancer, patients were required to be iso-
lated in a specialized hospital facility for a week to
ensm'e that the virus had completely cleared before
being allowed back into the general population.
Because of these limitations, there have been relative-
ly few trials with oncolytic therapy.
Even tn this early stage, oncolytic viral therapy has
demonstrated some success 46. Both a d e n o v i r u s and
herpes virus agents have o n g o i n g clinical trials for
intractable c a n c e r s (table 1). The most notable aden-
oviral therapy is the ONYX-015 viral therapy. ONYX-
015 is an a d e n o v i r u s that has been engineered to lack
Clhl Transl OncoL 2006;8(12):858-67
the viral EIB protein 47. Without this protein, the virus
is u n a b l e to replicate in cells with a n o r m a l p53 path-
way. In addition, the EIB protein is essential for RNA
export d u r i n g viral replication 4"s. Cancer cells often
have deficiencies in the p53 p a t h w a y due to m u t a -
tions and thus, allow ONYX-015 to replicate and lyse
the cells. C a n c e r cells also exhibit altered RNA export
m e c h a n i s m s that allow for the export of viral RNA
even in the a b s e n c e of the E1B protein ~'s. ONYX-015
is currently being tested as a preventative t r e a t m e n t
for p r e c a n c e r o u s state, as there are the p53 p a t h w a y
inactivating mutations, which will allow the oncolytic
a d e n o v i r u s to replicate and eliminate the cells before
they become c a n c e r o u s 49.
The second type of oncolytic virotherapy u n d e r g o i n g
clinical trials uses herpes simplex virus type 1 (HSV-
I). Two vectors, G207 and NVI020 are currently in
phase 1 and phase 11 trials for treatment of intractable
cancers. Mutations in several genes of these herpes
viruses e n s u r e that they replicate efficiently only in
cancerous cells. G207 is mutated so that it has attenu-
ated n e u r o v i r u l e n c e and c a n n o t replicate in n o n d i -
viding cells 5~ NVI020, a derivative originally used for
vaccine studies, has multiple mutations, i n c l u d i n g a
deletion in the t h y m i d i n e kinase gene u n d e r the con-
trol of the 14 p r o m o t e r 5~ These viral vectors have
two distinct cell killing m e c h a n i s m . The lyric phase of
the life cycle directly kills cells and the t h y m i d i n e ki-
nase that is expressed from the viral genes sensitizes
cells to gancielovir. These viral therapy vectors have
been used with great success in vitro and in model
a n i m a l s against a wide n u m b e r of solid cancers 51-55
Clinical trials using these vectors i n c l u d e a phase 1
trial of G207 for treatment of m a l i g n a n t glioma 54" and
a phase 1/11 trail of NVI020 for treatment ofcolorectal
cancer metastases to the liver 55 and for treatment of
glioblastoma 55.
FUTURE DIRECTIONS
Because viral gene therapy is not yet a mature tech-
nologT, there is plenty of room for improved treat-
ment vectors. In order for viriotherapy to be success-
fill, viral particle production rates in the infected
cancer cells. This may be difficult to achieve with
large established t u m o r s 5ti and may m e a n that virio-
therapy must be c o m b i n e d with an existing therapy,
such as surgery, to decrease the n u m b e r of c a n c e r
cells in the initial treatment. In addition, the most ef-
[~ctive treatment delivery method is yet to be deter-
mined. 111 p r e l i m i n a r y studies, systemic injection re-
quired a one-thousand-fold viral load compared to
that necessary to achieve results in i n t r a t u m o r a l l y in-
jection 57.
However, once these factors are overcome, there are
m a n y benefits to oncolytic therapy. The selective na-
ture of viriotherapy e n s u r e s that healthy tissue will
8~5
 MANCHEIqO-CORVO P, MARTiN-DUQUE P. VIRAL GENE THERAPY

TABLE 1. Examples of D e l e t i o n - m u t a n t - C R A d s

Characteristics in normal
Adenovirus Target Stage References
cells

dl1520 (Onyx-015) E1B-55K deletion Cells with mutant or absent Several clinical trials in (Bischoff et al., 1996;
prevents p53 binding p53 or that can complement a variety of cancers. Jiang et al., 2006;
and efficient late viral for late viral mRNA export. Best results observed in Khuri et al., 2000;
mRNA export. a Phase II trial for head O'Shea et al., 2004)
and neck cancer in
combination with
chemotherapy. Approved
for Phaselll trial in China.

ADD24 (dl922-947) E1A mutation on CR2
region prevents pRb
binding and efficient
induction of the cell cycle.
Cells with disrupted Phase I clinical trial in (Fueyo et al,, 2000;
pRb function. malignant gliomas; Phase I Heise et al., 2000)
clinical trial in ovarian cancer
with an infectivity-enhanced
version.

dl118 E1B-55k deletion prevents Cells with mutant of absent Pre-clinical. Effective in (Martfn-Duque et al.,
p53 binding. Increased p53. a panel of cancer cell lines 1999)
cytotoxicity due to including cervix, colon, breast
E1B-19k absence. and osteosarcoma.

diE1 bl 9k E1B-19K deletion
abrogates viral replication
due to increased toxicity.
Cells with altered apoptotic Pre-clinical. Effective in a (Liu et al., 2004)
pathways. panel of cancer cell lines
including breast, ovarian,
hepatic and pancreatic cancer
cells, among others.

dlVA-I VA-RNA I deletion causes Cells with Ras mutations Pre-clinical. Tested in pancreatic (Cascallo et al., 2003;
poor replication due to that revert PKR activation, cancer with Ras mutations and Wang et al., 2005)
PKR activation. or EBV associated tumours. Burkitt's lymphoma cells.

be m i n i m a l l y impacted. In addition, w h e n c o m b i n e d ACKNO~VLEDGEM ENTS

with cytotoxic gene expression, this t h e r a p y can affect
not only rapidly dividing cells, but those in the sur-
r o u n d i n g tissue m a k i n g the mic roenvi tom ent less fit-
vorable for c a n c e r growth. The c o m b i n a t i o n of" the
powerf"ul killing nature of these vectors c o m b i n e d
with the selectivity m a k e s them an exciting a v e n u e
for l o w e r i n g the n u m b e r of c a n c e r deaths.
Authors would like to a c k n o w l e d g e to Rodrigo Die-
guez by his contribution on the manuscript. Research
is s u p p o r t e d by Francisco de Vitoria University, Ra-
m 6 n y Cajal p r o g r a m fi'om the Ministery of Education
and Science and by Fondo de Investigaciones Sanita-
rias fi'om the spanish Ministery of Health.

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