Portada Simposios - Supplements - Haematologica

XLII Reunión Nacional de la AEHH y XVI Congreso de la SETH. Simposios
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lia. The expression of high levels of FVIII protein has
been traditionally difficult 5 . As a result, efforts to deliver
FVIII have historically been preceded by attempts
to deliver FIX.
There are two distinct approaches to gene therapy,
depending on whether the patient is transplanted
with recombinant cells secreting a therapeutic product
(ex vivo), or injected directly with DNA, such as
a viral vectors that target host cells (in vivo).
In vivo gene therapy
Viral vectors
The potential of viral vectors to efficiently target
and infect mammalian cells can be exploited to deliver
a therapeutic gene. A number of viral vectors
have been used to deliver FVIII and FIX.
Retroviral vectors are attractive because they integrate
within the genome of target cells. Therefore,
transgene expression is likely to be sustained and will
not be lost upon cell division. Retroviral vectors have
been used to deliver FVIII and FIX in mice, dogs and
rabbits 5 . More recently, physiological levels of hFVIII
have been sustained in newborn hemophilic mice injected
IV with a new generation of retroviral vectors 6 .
However, not all treated mice were tolerized to the
foreign human protein, and some mice developed
inhibitors 6 . Chiron began in June 1999 a human trial
based on the IV injection of retrovirus in severe haemophilia
A patients. Initial results of this trial are not
yet available. One potential concern of this approach
is the possibility that integration of retrovirus
can lead to genomic reorganization, such as oncogene
activation. In addition, the spread of vector
could potentially affect the gonads. In this event, the
consequences for the patient and his progeny must
be seriously considered.
Adenovirus is a benign virus that can infect dividing
as well as non-dividing cells. This is an important
advance, since cells in potential target organs for
gene therapy such as liver or brain are not noticeably
proliferative. In addition, adenoviral vectors can
achieve extremely high levels of transgene expression.
In fact, adenoviral vectors have delivered supraphysiological
levels of FVIII and FIX in animal models of
haemophilia 5 . However, the highly antigenic nature of
adenovirus elicits a strong cellular immune response
against the infected cells. In addition, adenoviral vectors
do not integrate into the host’ genome, but rather
stays episomal. As a result, adenovirus genome
is diluted upon cell division, and so too is the therapeutic
gene. Therefore, transgene delivery from
adenovirus is typically short-lived. Furthermore, repeat
adenovirus treatments are consistently ineffective
due to its highly antigenic nature. In order to reduce
vector antigenicity, recent developments have
been directed at eliminating most viral genes in adenoviral
vectors. Scientists at Baxter have engineered a
mini-adenoviral vector devoid of all viral genes. Following
intravenous injection, this vector can sustain
physiological levels of FVIII in some, but not all, hemophilic
mice for at least 1 year 7 . In collaboration
with Baxter, GenStar Therapeutics Corp. intend to
initiate human trials shortly. However, the recent death
of an 18 year-old patient suffering from OTC deficiency
following an adenovirus treatment 8 has raised
serious concerns about the immune responses to
adenovirus, and the safety of this virus. In particular,
it is worth to point out that vectors delivered intravenously
mainly target the liver. This fact should be considered
when designing a therapy for severe haemophiliacs,
many of whom have liver damage following
infections suffered from the use of tainted blood.
Adeno-associated virus (AAV) is a small benign parvovirus
that requires a helper virus, such as adenovirus,
for infection. Just like adenovirus, AAV can also
infect dividing and non-dividing cells. Following infection
of human cells, wild type AAV integrates specifically
in chromosome 19. This peculiarity of AAV
could eliminate the safety concerns regarding oncogene
activation associated to retrovirus. As opposed
to adenovirus, AAV is not very antigenic. As a result,
transgene expression can be sustained long-term. Indeed,
injection of AAV into the liver via the portal vein
lead to persistent delivery of curative levels of FIX
(2 g/ml, or ∼i40 % of physiological levels in humans)
in hemophilic mice for more than a year 9 . No antibodies
to hFIX were detected in the treated mice. Interestingly,
when the same vector was injected intramuscularly
into hemophilic mice, FIX levels were lower
(200-300 ng/ml). More importantly, these mice
developed antibodies to FIX 10 . These findings highlight
the fact that the mode of transgene delivery
plays a critical role in the presentation of a foreign or
novel antigen to the immune system. This has wide
implications for all gene therapy approaches. Intramuscular
and intraportal injection of AAV has also
been successfully used to deliver sustained levels of
FIX in hemophilic dogs 11,12 . The dose achieved was,
however, lower than that detected in hemophilic mice
(∼i1-3 % of physiological levels in humans). A mild
antibody titer was observed in some dogs, although
antigen delivery was not compromised.
Based on these encouraging pre-clinical results, a
human trial for haemophilia B began in 1999. In this
trial, patients are treated once with AAV as multiple intramuscular
injections. Initial results of the first three
patients treated with a low AAV dose indicate that the
procedure is safe and well tolerated by patients 13 . In
addition, modest but nevertheless detectable levels of
FIX were observed in at least one patient. More importantly,
a reduction in the FIX infusion regimen of
the treated patients was observed, clearly indicating a
clinical benefit of the procedure. Indeed, AAV is arguably
the most promising vector to date. The small
size of AAV has prevented the use of AAV for the delivery
of FVIII. The size of AAV is 4.7 kb, and the size of
the complete FVIII cDNA is ∼i7 kb, and 4.5 kb without