Advanced therapies for hemophilia: Reality or fantasy?

Article (PDF Available)inExpert Review of Hematology 5(3):245-7 · June 2012with26 Reads
Impact Factor: 2.07 · DOI: 10.1586/ehm.12.25 · Source: PubMed

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Available from: Antonio Liras, Mar 07, 2014
ISSN 1747-4086
© 2012 Expert Reviews Ltd
Future therapeutic products based on
advanced therapies such as gene and cell
therapy and tissue engineering or induced
pluripotent stem cell technology may offer
innumerable potential clinical applications
for the treatment of several monogenic
disorders including hemophilia. Although
hemophilia is particularly amenable to
treatment with these techniques given its
monogenic nature and the lower levels of
deficient coagulation factor required to
achieve a moderate phenotype, research
in the hemophilia field is still at a teething
stage and further work must be undertaken
to determine whether advanced therapies
can be safely applied to this patient popula-
tion, which presents specific clinical char-
acteristics. There is much reason for opti-
mism, but caution is imperative in order not
to raise false expectations in our patients.
The development of biotechnology
has resulted in the emergence of new
therapies that are bound to change medi-
cal practice. Advanced gene-, cell- and
tissue-based therapies (gene therapy, cell
therapy and regenerative medicine
constitute new strategies for the treat-
ment of some diseases [1] . Their purpose
is either the regeneration of tissues or the
restoration of function. Cell therapy con-
sists of the transplantation of living cells
into an organism in order to repair tissue
or restore an absent or deficient function.
Gene therapy, in turn, consists of trans-
plantation of genetically modified cells so
that they may produce a deficient protein.
Cells are useful in these therapies
because of their ability to differentiate into
the specific cell lines required for restoring
a given type of tissue. Nonetheless, only
20% of stem cell-related studies in the
literature constitute a genuine advance-
ment in scientific knowledge. This can
be attributed to the high cost of this kind
of research and to the multiplicity of yet-
to-be-resolved issues such as the need to
develop new guidelines on best practices
in cell culture and cell transplantation
procedures, and guarantee the genetic
stability of stem cells before and after
transplantation, their quantity and quality
when used therapeutically and their safety,
specifically with respect to the absence of
Current treatment of hemophilia is
based on the replacement of deficient
coagulation factors by prophylactic or
on-demand intravenous administration.
Given the monogenic nature of hemo-
philia and the low coagulation factor levels
required to convert a severe into a mild or
moderate phenotype, it is thought that the
disease may in future be particularly ame-
nable to treatment with advanced therapies
[35]. Most clinical and preclinical studies
conducted to date on the effects of cell
therapy and gene therapy on hemophilia,
using both viral and nonviral vectors, have
shown no adverse effects, although the
immune response against the vectors’ viral
envelopes and the transgenes encoded con-
stitute the limit to the clinical application
of these therapies.
Gene therapy strategies for hemophilia
have been based on the use of lentiviral
and adeno-associated virus (AAV) vectors,
adult stem cells and autologous fibroblasts,
platelets and hematopoietic stem cells.
Advanced therapies for hemophilia: reality
or fantasy?
Expert Rev. Hematol. 5(3), 245–247 (2012)
Keywords: advanced therapies • cell therapy • gene therapy • hemophilia
There is much reason for optimism, but caution is imperative in
order not to raise false expectations in our patients.
Expert Review of Hematology
© 2012 Expert Reviews Ltd
Advanced therapies for hemophilia: reality or fantasy?
Expert Rev. Hematol.
Antonio Liras
Department of Physiology,
Biological Sciences School,
Complutense University of
Madrid, Spain
Royal Foundation “Victoria
Eugenia” of Haemophilia, Spain
Tel.: +34 630 068 924
Fax: +34 916 379 651
For reprint orders, please contact
Page 1
Expert Rev. Hematol. 5(3), (2012)
Nonviral gene transfer and chimerical oligonucleotide-based
mutation repair have also been used. Cell therapy for hemophilia
is based, mainly, on the transplantation of healthy cells to repair
or replace deficient functions, for example the transplantation of
liver sinusoidal endothelial cells or endothelial progenitor cells
derived from induced pluripotent stem cells. Of particular interest
in the field of advanced therapies are the results obtained by
High et al. who used zinc finger nucleases and adeno-associated
vectors to correct hemophilia B mutations through the ‘editing’
of DNA-mutated sequences [6]. Although in this case factor IX
(FIX) expression is only 5% of normal levels, the advantage of
this strategy is that it allows strict control of the integration of
normal sequences into DNA, thus preventing the development
of insertional mutagenesis-induced tumors.
In our laboratory, we have used nucleofection-based nonviral
gene transfer to promote the expression of human FIX in adult
adipose tissue-derived mesenchymal stem cells [7] . Despite the fact
that the expression efficacy of the nonviral method is lower than
that obtained with viral vectors, the former provides higher safety
levels, with the additional advantage that 5% of the coagulation
factor level is enough to transform a severe hemophilia phenotype
into a moderate one. The most significant problems that remain
to be addressed are related to increasing the efficacy of factor
expression levels and maintaining them at a constant level in the
long term, and control of the immune response to vectors and
Nathwani et al. have recently completed a clinical trial in
patients with hemophilia B. The trial included patients with
severe hemophilia B (<1% FIX) who were injected with a
serotype-8-pseudotyped, self-complementary AAV, that expresses
FIX and can efficiently transduce hepatocytes [8] . This is a more
efficient vector as it obviates the need for second-strand synthesis
or re-annealing of positive and negative AAV strands to generate
transcription-competent dsDNA templates. The results showed
that patients expressed between 3 and 11% of normal FIX lev-
els. Another encouraging finding was that no inhibitors (anti-
FIX antibodies) were detected. These results must be considered
taking into account, first of all, that the expression of FIX cor-
responds to a mild-to-moderate phenotype of the disease and,
second, that concomitant glucocorticoid treatment is required
in order to prevent immune rejection and an elevation of liver
The results obtained to date constitute the beginning of the
future application of advanced therapies to the treatment of
hemophilia. The number of patients included in the clinical tri-
als on advanced therapies conducted so far, including the one
by Nathwani et al., has been very low. In addition, results have
been highly variable. Although the clinical trial by Nathwani
et al. is the first study to show a substantial expression of FIX in
humans, immune-mediated clearance of AAV-transduced hepato-
cytes remains a concern. Specifically, a patient who had received
a high dose of the vector developed Grade III liver toxicity, which
was related to the vector itself. The same patient also showed an
increase in plasma transaminase levels and a concomitant decrease
in FIX levels. All of these findings were related to the presence of
AAV8 caspid-specific T cells. An important question that remains
to be resolved is that of the potential relationship between liver
toxicity and the immune response it generates. Additionally, there
are still a few things to be clarified about the study by Nathwani
et al., such as the case of two patients who were administered a
mild dose of the vector and, although they did not present with
liver toxicity, they did exhibit an immune response to the vector
capsid. In order to resolve this, future clinical trials must include
a larger number of patients. Another important consideration is
that even if the expression levels obtained are substantial, they
are not sufficient to consider healing of hemophilia and to assume
that bleeding episodes further to trauma or injury will be pre-
vented. Future efforts must be aimed at improving the design of
the vectors as the truncated liver-specific promoter used in this
clinical trial to accommodate the FIX gene in the self-comple-
mentary AAV backbone to make it more efficient. This could
allow the use of lower vector doses and the reduction of potential
vector-dependent liver toxicity. Another drawback is the problem
of transient transgene expression.
There is much reason for optimism, but we need to act cau-
tiously so as not to create premature expectations in our patients.
It is almost two decades since the first reports on the benefits
of gene therapy for the treatment of hemophilia came to light.
In those days, prominent experts envisioned that the cure of
this disease could become an achievable goal by the first dec-
ade of the 21st century [9]. These predictions fueled the hopes
of both hemophilic patients and the physicians treating them,
but unfortunately they were in for a disappointment. Problems
started to emerge, especially in the field of biosafety, as early
as the 1990s when the first clinical trials on gene therapy were
launched. Although significant progress has been made since
then in terms of the design of transfer vectors, some of the
drawbacks associated with the technique have not as yet been
fully overcome (host immunity, insertional mutagenesis, efficacy
and expression time, clinical trial recruitment and large-scale
vector production).
The first question that must be asked is whether the time and
financial investment required to establish advanced therapy pro-
tocols, that may in future be applied to the treatment of this
pathology is justified. Although it must be admitted that current
treatment of hemophilia is optimal, the answer to the question is
in the afrmative since hemophilia is a chronic condition and cur-
rent high-frequency – especially in prophylaxis – treatment proto-
cols apart from being extremely costly, could result in devastating
pathogen-induced infections. The second question is whether
advanced therapies are at all feasible. In this regard, hemophilia
...research in the hemophilia field is still at a
teething stage and further work must be
undertaken to determine whether advanced
therapies can be safely applied to this patient
population, which presents specific clinical
Page 2
is considered an optimal candidate for such treatments as it is a
monogenic disease; the expression of low levels of the coagulation
factor (1–5%) can achieve a moderate phenotype; a large variety
of applicable target cells exist; there is no need to regulate factor
expression; and a large amount of animal models are available
for experimentation.
Other more general questions that may be raised include
whether it will be possible to extrapolate the safety- and expression
level-related outcomes obtained in animals to human beings,
whether the combination of cell therapy/gene therapy with the
use of mesenchymal stem cells will be the most efficient tool and
whether protocols will have to be restricted to AAV and nonviral
The problems to be addressed in the future are the immuno-
genicity and biosafety of the therapies as well as the maintenance
of the levels and time of factor expression. Moreover, although
most research is currently focused on viral vectors, nonviral
vectors should also be taken into consideration.
At any event, the main criterion to be considered should be the
ratio between efficacy and safety, taking into account that this
is a highly sensitive issue for both patients and physicians given
the special immunologic situation of the patients and the lethal
consequences that viral infections (HIV/HCV) have had on the
hemophilic population in the past. Would it be wise to forego
a set of stringent expression requirements in return for greater
safety [10] ?
Furthermore, caution must be exercised when bringing to
light the results of any studies that may be conducted in the
field. With regard to the clinical trial by Nathwani et al., in
which severe hemophilia B patients were subjected to perfusion
of a serotype-8-pseudotyped, self-complementary AAV that
expresses FIX, its results must be examined taking into account
first and foremost that the expression levels obtained correspond
to a moderate-to-mild phenotype of the disease. Moreover, it
must be remembered that concomitant gluco-corticoid therapy
is indispensable to prevent immunological rejection and normal-
ize liver transaminase levels. In addition, the virus is hepatotoxic,
the number of patients is limited and results are highly variable.
Although the results reported by Nathwani et al. are a break-
through, the significant difficulties posed by the different strate-
gies of gene and cell therapy used so far require extreme prudence
and objectivity to be exercised before any conclusions are drawn.
Statements in the study such as “If further studies determine
that this approach is safe, it may replace the expensive protein
therapy currently used for patients with hemophilia B,” or in the
accompanying editorial [11] , are rather unwarranted, because it
may be years or even decades before this technology can be used
in clinical practice.
False expectations with respect to advanced therapies that are
only in the initial phases as potentially highly promising therapeu-
tic strategies should not be raised. In the longer term, after over-
coming the challenges mentioned above, advanced gene- and cell-
based therapies might become a plausible alternative for patients
with hemophilia. Optimism is in order, but fantasy is best avoided.
Financial & competing interests disclosure
The author was supported by funding from a grant from the Royal Foundation
“Victoria Eugenia” of Haemophilia. The author is also Principal Researcher
in a preclinical project, but not a clinical trial, with adipose mesenchymal stem
cells and gene/cell therapy protocols for treatment of hemophilia. The author
has no other relevant affiliations or financial involvement with any organiza-
tion or entity with a financial interest in or financial conflict with the subject
matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
1 Tirino V, Papaccio G. A new, most likely
unusual approach is crucial and upcoming
for the use of stem cells in regenerative
medicine. Front. Physiol. 2, 1–2 (2012).
2 Liras A. Future research and therapeutic
applications of human stem cells: general,
regulatory, and bioethical aspects. J. Transl.
Med. 8, 131 (2010).
3 Liras A. Induced human pluripotent
stem cells and advanced therapies:
future perspectives for the treatment of
haemophilia? Thromb. Res. 128(1), 8–13
4 Vandendriessche T, Chuah MK. Clinical
progress in gene therapy: sustained partial
correction of the bleeding disorder in
patients suffering from severe hemophilia B.
Hum. Gene Ther. 23(1), 4–6 (2012).
5 Liras A. Gene therapy for haemophilia:
the end of a ‘royal pathology’ in the third
millennium? Haemophilia 7(5), 441–445
6 Li H, Haurigot V, Doyon Y et al. In vivo
genome editing restores haemostasis in
a mouse model of haemophilia. Nature
475(7355), 217–221 (2011).
7 Olmedillas S, García-Arranz M, Vega L,
García-Gómez I, García-Olmo D, Liras A.
Non-viral gene transfer to human adipose-
derived stem cells for FIX secretion. Presented
at: 4th National Congress of Spanish Society of
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21–23 September 2011.
8 Nathwani AC, Tuddenham EG, Rangarajan
S et al. Adenovirus-associated virus vector-
mediated gene transfer in hemophilia B.
N. Engl. J. Med. 365(25), 2357–2365 (2011).
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101 European Medicines Agency. Advanced
There is much reason for optimism, but we need
to act cautiously so as not to create premature
expectations in our patients.
Advanced therapies for hemophilia: reality or fantasy?
Page 3
    • "Factor administration increases patients' quality of life and reduces the long-term surgical cost associated with hemophilia (Srivastava et al., 2013). In the longer term, advanced therapies (Liras, Segovia & Gaban, 2012; Liras, 2012), gene therapy (using adeno-associated, lentiviral vectors or non-viral vectors) (Walsh & Batt, 2013) and cell therapy (through the use of adult stem cells or induced pluripotent stem cells) (Miao, 2012), are likely to become the treatment of choice for the disease. Hemophilia is considered an optimal candidate for these strategies given its monogenic origin and because a slight increase in factor levels—over 5%—can convert a severe into a mild or moderate hemophilia phenotype with a significant reduction in the risk of hemorrhagic diathesis. "
    [Show abstract] [Hide abstract] ABSTRACT: Hemophilia is a rare recessive X-linked disease characterized by a deficiency of coagulation factor VIII or factor IX. Its current treatment is merely palliative. Advanced therapies are likely to become the treatment of choice for the disease as they could provide a curative treatment. Methods. The present study looks into the use of a safe non-viral transfection method based on nucleofection to express and secrete human clotting factor IX (hFIX) where human adipose tissue derived mesenchymal stem cells were used as target cells in vitro studies and NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice were used to analyze factor IX expression in vivo studies. Previously, acute liver injury was induced by an injected intraperitoneal dose of 500 mg/kg body weight of acetaminophen. Results. Nucleofection showed a percentage of positive cells ranging between 30.7% and 41.9% and a cell viability rate of 29.8%, and cells were shown to secrete amounts of hFIX between 36.8 and 71.9 ng/mL. hFIX levels in the blood of NSG mice injected with ASCs transfected with this vector, were 2.7 ng/mL 48 h after injection. Expression and secretion of hFIX were achieved both in vitro cell culture media and in vivo in the plasma of mice treated with the transfected ASCs. Such cells are capable of eventually migrating to a previously damaged target tissue (the liver) where they secrete hFIX, releasing it to the bloodstream over a period of at least five days from administration. Conclusions. The results obtained in the present study may form a preliminary basis for the establishment of a future ex vivo non-viral gene/cellular safe therapy protocol that may eventually contribute to advancing the treatment of hemophilia.
    Full-text · Article · Apr 2016
  • [Show abstract] [Hide abstract] ABSTRACT: Introduction: In hemophilia, advanced therapies are warranted from a conceptual and methodological standpoint. Current advanced therapy strategies are centered on the use of adeno-associated viral vectors, although problems related to immunogenicity and hepatotoxicity still remain. Areas covered: Future clinical trials will have to scrupulously observe international bioethical standards in terms of patient selection, particularly children. Patient recruitment rates are likely to remain low due to the stringent exclusion criteria usually imposed on the trial population regarding their hepatic and immunological markers and the presence of viral coinfection; and to the existence of an optimal palliative treatment. Expert opinion: Accordingly, the results obtained are likely to be of low statistical significance, which could hinder their application to clinical practice. Another important issue is the degree to which society embraces these new emerging therapies. The unfamiliarity of society with these new methods, together with the many unresolved questions about them that remain, may threaten their acceptance not only by society at large but also by health-care professionals, which would limit their translational application to clinical practice.
    Full-text · Article · Mar 2015

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