Advanced therapies for the treatment of hemophilia: future perspectives


Advanced therapies for the treatment of hemophilia

In the future, the different types of advanced therapies such as gene therapy, cell therapy and tissue engineering, as well as the more recently developed induced pluripotent stem cells (iPSC) technology, may offer innumerable clinical applications for the treatment of certain monogenic diseases including hemophilia.

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True as it is that hemophilia is well suited to be treated by advanced therapy protocols on account of its monogenic nature and of the fact that a modest increase in coagulation factor levels is enough to convert a severe into a moderate phenotype, it cannot be forgotten that research in this field is still at an early stage and substantial efforts will have to be made before such therapies can be made readily available to the patients, particularly in terms of safety. Safety considerations will have to be taken very seriously, notably in this group of patients who present with specific clinical characteristics that more often than not are the result of their past and present-day treatment. These characteristics include the presence of inhibitors or the predisposition to develop them, the specific immunological status of these patients and the presence of viral co-infections (HIV/HCV) [28]. For these reasons, although optimism is certainly in order, caution is of the essence to avoid raising false expectations in both physicians and patients alike.

Gene therapy strategies

Gene therapy consists of transplantation of genetically modified cells so that they may produce a functional protein, and cell therapy in the transplantation of living cells into an organism in order to repair tissue or restore a deficient function. Both strategies are based on the use of stem cells given their indefinite capacity to renew themselves and differentiate to become cells of several specific cell lines. All these are necessary conditions for their clinical application [29].

The most significant breakthroughs in the field of advanced therapies and hemophilia are chiefly related to both preclinical and clinical trials in the fields of gene therapy (through the use of viral and non-viral vectors) and cell therapy (using several types of target cell) (Table 3). Thus hemophilia reveals itself as a disease that is highly amenable to be treated by gene therapy [30, 31, 32, 33] by means of lentiviral and adeno-associated vectors used in adult stem cells and autologous fibroblasts, platelets or hematopoietic stem cells; and of the transfer of non-viral vectors and repair of mutations by chimeric oligonucleotides. The studies published so far have, in the most part, not reported any adverse event resulting from the application of such strategies in the clinical trials performed in terms of an immune-mediated transgene rejection (factor VIII or IX expression) although factors such as innate cellular T cell toxicity to adeno-associated capsid protein and the low efficacy obtained by non-viral vectors are impeding and limiting their success [34].
Table 3

Preclinical studies and clinical trials on gene- and cell therapy for hemophilia

Authors [Reference]

Vector or target (tissue) cells

Coagulation factor expressed

Expression level (%)

Gene therapy (Preclinical studies)

Jeon et al. Ref.[35]




Brown et al.Ref.[34]




Ramezani et al.Ref.[36]




Matsui. Ref.[37]




Montgomery and Shi. Ref.[40]




Gene therapy (Clinical trials)

Nathwani et al.Ref.[38]

AAV (Immunosuppressive therapy)



Buchlis et al.Ref.[39]



FIX RNA expression and AAV DNA persistence (<1% FIX)

Cell therapy (Preclinical studies)

Aronovich et al.Ref.[43]

Embryonic day 42 spleen tissue



Follenzi et al.Ref.[44]

Liver sinusoidal endothelial cells



Follenzi et al.Ref.[45]

Kupffer cells. Bone marrow-derived mesenchymal stromal cells



Xu et al. Ref. [46]

iPSCs from tail-tip fibroblasts and their differentiation into endothelial cells and their precursors



Yudav et al.Ref. [47]

Transdifferentiation of iPSC-derived endothelial progenitor cells into hepatocytes



LVV, lentiviral vector; AAV, adeno-associated vector; FIX, factor IX.

Brown et al.were the first to use lentiviral vectors for treatment of hemophilia B. Using a lentiviral vector containing a target sequence for the hematopoietic-specific microRNA, miR-142-3p, they obtained 10% factor IX activity with no anti-FIX antibodies in hemophilia B mice at over 280 days after injection. Since those results were published, use of lentiviral vectors has not ceased to grow. Thus, Jeon et al transduced this type of viral vector into skeletal muscle to increase factor VIII expression. Factor VIII plasma levels at one week post-injection were 5.19 ng/mL vs 0.21 ng/mL in control rats, with those levels staying constant over 4 weeks with a single dose of the vector. More recently, and also using lentiviral vectors, Ramezani et al., adapted a nonmyeloablative conditioning regimen and directed factor VIII protein synthesis to B lineage cells using an insulated lentiviral vector containing an immunoglobulin heavy chain enhancer-promoter. Transplantation of lentiviral vector-modified hematopoietic stem cell resulted in an increase in factor VIII plasma levels for 6 months, with a low immune response against the protein expressed and a correction of the hemophilic phenotype in the transplanted mice.

Very recently, Matsui et al., established a gene therapy strategy using autologous circulating endothelial progenitor cells transfected with lentiviral vectors containing a canine FVIII transgene. When implanted subcutaneously in a soluble basement membrane scaffold, these cells produced a long-term FVIII expression over 6 months, and resulted in effective prophylaxis against bleeding.

Ward et al., have directly compared, using lentiviral vectors, FVIII expression from FVIII-constructs containing various B domains from non–codon-optimized and codon-optimized cDNA sequences without the confounding effect of variable immune responses against human FVIII, neo epitopes and the Fugu B domain. A dramatic increase in the observed level of secreted FVIII from codon optimized cDNA sequences was obtained. These results are in contrast to transient FVIII expression levels obtained from another many previous approaches.

Gene therapy studies conducted in hemophilic patients showed that use of adeno-associated vectors currently constitutes the most promising option given the high safety profile of such vectors, although they are not exempt from immune response-related problems. Efforts are nowadays directed at reducing the incidence of immune rejection and increasing the efficacy and length of expression. Several studies have been published in an attempt to optimize the use of viral vectors. Thus, Nathwani et al., completed a pioneering clinical trial in patients with severe hemophilia B (<1% FIX). Patients were perfused with a dose of a serotype-8-pseudotyped, self-complementary adeno-associated vector that expressed factor IX and could efficiently transduce hepatocytes. Their results showed that factor IX expression ranged between 2 and 11% of normal values. Significant as they may seem, these results must be considered with caution as the expression levels achieved rather than normalize the patient’s phenotype convert it to a mild-to-moderate form. Also treatment with glucocorticoids may be necessary to prevent immune rejection and increase the duration of transgene expression. Due account must also be taken of the fact that the adeno-associated vector has the potential to induce hepatotoxicity. For all these reasons, these undoubtedly encouraging results can only be considered a first step in the development of safe and effective advanced therapies for the treatment of hemophilia.

A recent study  reported on the persistent long-term expression of factor IX in parenteral administration of an adeno-associated viral vector in muscle tissue. The authors show that adeno-associated serotype-2-mediated gene transfer to human skeletal muscle persists and is transcriptionally and translationally active for a period of up to 10 years. This is the longest reported transgene expression to date.

A new alternative that has been proposed in connection with gene therapy strategies for hemophilia is the use of platelet targeting as a mechanism of drug delivery . Such a strategy could play an efficient clinical role in the treatment of hemophilia and other hemostatic disorders given that it would allow local release of factor VIII and IX at the site of the bleeding-induced damage and, at the same time, protect such factors from the effect of the inhibitors potentially present in plasma.

Non-viral strategies also play an important role in this area as they constitute a safe alternative for the future in the face of the limitations that have so far been associated with viral vectors in terms of their biosafety and potential clinical application.

Thus, Sivalingam et al., evaluated the genotoxic potential of phiC31 bacteriophage integrase-mediated transgene integration in cord-lining epithelial cells cultured from the human umbilical cord. This non-viral strategy has made it possible to obtain stable factor VIII secretion in vitro. Xenoimplantation of these protein-secreting cell lines into immunocompetent hemophilic mice corrects the severe form of the disease.

Our laboratory has advanced the use of nucleofection as a non-viral transfection method to obtain factor IX expression and secretion in adult adipose tissue-derived mesenchymal stem cells . Although it is certainly true that expression efficacy with these types of protocols is lower than when viral vectors are used, it must be remembered that achieving a factor plasma level of at least 5% can transform a severe into a mild phenotype. In addition, non-viral vectors provide higher safety levels than viral ones.

Cell therapy strategies

The use of cell therapy in the treatment of hemophilia has to date consisted mainly in the transplantation of healthy cells in an attempt to repair or replace a coagulation factor deficiency. These procedures have been conducted mainly with adult stem cells and, more recently, with progenitor cells partially differentiated from iPSCs, albeit in most cases the mechanisms by which transplanted cells (to a greater or lesser extent) engraft and go on to proliferate and function remain unknown.

Aronovich et al., have shown that transplantation of embryonic day 42 spleen tissue in immunocompetent mice with hemophilia A attenuates the severity of the disease in the 2–3 months after the procedure. These results would seem to indicate that transplantation of a fetal spleen —obtained from a developmental stage prior to the appearance of T-cells— may potentially be used to treat some genetic disorders. For their part, Follenzi et al. reported that once liver sinusoidal endothelial cells were transplanted and successfully engrafted into mice with hemophilia A, they were seen to proliferate and partially replace some areas of the hepatic endothelium. This resulted in a restoration of factor VIII plasma levels and in the correction of the bleeding phenotype. More recently, this same team  demonstrated that transplantation of healthy mouse Kupffer cells (liver macrophage/mononuclear cells), which predominantly originate from bone marrow, or of healthy bone marrow-derived mesenchymal stromal cells, can correct the phenotype of hemophilic mice and restore factor VIII levels in plasma.

As far as the use of iPSCs is concerned, the first paper was published by Xu et al. who reported on the generation of murine iPSCs from tail-tip fibroblasts and their differentiation into endothelial cells and their precursors. These iPSC-derived cells express specific membrane markers such as CD31, CD34 and Flk1, as well as factor VIII. Following transplantation of these cells into mice with hemophilia A, the latter survived the tail-clip bleeding assay by over 3 months and their factor VIII plasma levels increased to 8%-12%. Yadav et al., have studied the transdifferentiation of iPSC-derived endothelial progenitor cells into hepatocytes (primary cells of FVIII synthesis). These cells were injected into the liver parenchyma where they integrated functionally and made correction of the possible hemophilic phenotype. High levels of FVIII mRNA were detected in the spleen, heart, and kidney tissues of injected animals with no induction of tumors or any other adverse events in the long-term. Alipio et al. for their part also reported on the generation of factor VIII in a hemophilic murine model one year after transplantation of iPSC-derived endothelial cells.

Advanced therapies in the hemophilic arthropathy

Lastly, it is important to consider the potential application of advanced therapies in the palliative treatment of the articular consequences of hemophilic arthropathy. Although adequate treatment is currently available for hemophilia, which is specifically efficient regarding the negative consequences of hemophilic arthropathy, it cannot be forgotten that only 25% of hemophiliacs, most of them living in developed countries, can benefit from such treatment. In the rest of the world, hemophilic arthropathy and its disabling sequelae are the norm. But even in the developed world many patients still present moderate or severe hemophilic arthropathy on account of the fact that they either developed inhibitors or started being treated a few decades ago when present-day therapies were still unavailable.

Against this background, advanced therapies may constitute a solution of these patients . Chondrocyte implantation and cell therapy using bioreactors, growth factors, mesenchymal stem cells and genetically modified cells may be used as an adjunct or even as an alternative to the current approaches (bone marrow stimulation, osteochondral autograft or allograft transplantation) for the repair of chondral damage in advanced arthropathic disease.

Mesenchymal stem cells appear hold great promise for chondral repair given their high differentiation ability and their proven therapeutic effects . Implantation of autologous chondrocytes or mesenchymal stem cells was up to now able to address only highly localized chondral lesions, and the use of bioreactors and growth factors, which stimulate cartilage formation, may optimize such strategies.


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