Mesenchymal stem cell treatment for hemophilia: a review …

Summary Top of page Summary Introduction Extracellular vesicles produced by stem cells What we have learned from mature liver cell transplantation in humans? MSCs in human clinical applications Disclosure of Conflict of Interests References

Hemophilia remains a non-curative disease, and patients are constrained to undergo repeated injections of clotting factors. In contrast, the sustained production of endogenous factors VIII (FVIII) or IX (FIX) by the patient's own cells could represent a curative treatment. Gene therapy has thus provided new hope for these patients. However, the issues surrounding the durability of expression and immune responses against gene transfer vectors remain. Cell therapy, involving stem cells expanded invitro, can provide de novo protein synthesis and, if implanted successfully, could induce a steady-state production of low quantities of factors, which may keep the patient above the level required to prevent spontaneous bleeding. Liver-derived stem cells are already being assessed in clinical trials for inborn errors of metabolism and, in view of their capacity to produce FVIII and FIX in cell culture, they are now also being considered for clinical application in hemophilia patients.

Hemophilia A is the most common severe inherited bleeding disorder, affecting 1 in 5000 male births. This pathology exhibits different phenotypic expressions depending on its associated factor VIII (FVIII) plasma levels, resulting in severe (<1%), moderate (15%), or mild (630%) forms of expression [1]. This disease originates from an inherited deficiency or dysfunction in the procoagulant FVIII, a crucial element of the intrinsic pathway of blood coagulation involved in the conversion of factor X to Xa [2]. In severe cases, FVIII deficiency leads to spontaneous bleeding and internal hemorrhage that can cause disability and even death, if left untreated [3].

There is currently no cure for hemophilia A. The rationale behind replacement treatment is to sufficiently increase concentrations of the missing factor to arrest spontaneous and traumatic bleeds. Plasma-derived products became available in the 1970s, proving effective in controlling bleeding episodes through the development of home-therapy programs. However, concentrates derived from pooled plasma were contaminated with HIV and the hepatitis B or C virus, causing post-transfusion hepatitis and immunodeficiency in almost all hemophilia patients who received these concentrates. In 1984, the FVIII gene was successfully cloned, enabling the production of recombinant human FVIII (rFVIII) using mammalian cell cultures, and the first rFVIII went on the market in 1992 [4].

The introduction of rFVIII revolutionized hemophilia patient management by providing an effective and safe treatment of bleeding episodes. Nevertheless, there remain several issues concerning FVIII replacement therapy that have yet to be resolved. The primary complications are as follows: the short half-life of replacement products, necessitating frequent intravenous infusions; the immunogenicity of FVIII concentrates; and the affordability and availability of FVIII products [5].

During the last decades, several attempts have been made to develop a long-term cure, such as gene and cell therapy. Hemophilia A is a perfect candidate for gene therapy, given its monogenetic nature that can potentially be cured by continuous endogenous FVIII expression. For hemophilia treatment, by increasing circulating clotting factor levels to above 1% of normal, it may be possible to obtain a prophylactic therapeutic effect, thereby reducing risks of both mortality and morbidity.

If gene therapy is able to slightly increase clotting factor levels, it could significantly improve the clinical phenotype [6]. Recently, hemophilia B gene therapy has achieved promising outcomes in human clinical trials [7]. A key advantage of the development of gene therapy strategies for hemophilia B is the relatively small size of the cDNA of FIX, measuring approximately 1.4 kB of the coding sequence. This renders it amenable to insertion into different gene transfer vectors and enables the addition of numerous transcriptional regulatory elements to both improve and restrict transgene expression in selected cell types. The cDNA of FVIIIis much larger than that ofFIX (>8kB) and cannot be as readily accommodated in gene transfer vectors. Several strategies have previously been attempted to overcome this difficulty, by either deleting the B-domain or using two viral vectors [8].

Moreover, the main complication of viral vector delivery of clotting factor transgenes is the host immune responses [9]. A pre-existing immune response against capsid proteins is one of the criteria excluding hemophilia patients from gene therapy. Approximately 40% of the adult human population possess neutralizing antibodies against the adeno-associated virus (AAV)-2 [10], and these antibodies can cross-neutralize other AAV serotypes. In addition, patients receiving systemic viral vector administration develop a postgene therapy immune response. This response was shown to be associated with the destruction of cells expressing viral proteins after transduction, thereby decreasing the gene transfer's efficacy, along with the development of neutralizing antibodies against the viral vector employed as therapeutic agent, therefore preventing the possibility of vector re-administration.

Orthotopic liver transplantation has proven effective in correcting the hemophilic phenotype in hemophilia patients with decompensated hepatitis C (HCV)-cirrhosis [11]. This suggests that the liver plays a central role in producing blood-clotting factors like FVIII. The hepatic cellular compartments that produce FVIII are primarily composed of liver sinusoidal endothelial cells (LSEC) [12, 13], although earlier evidence has suggested hepatocytes to be instrumental in FVIII expression [14]. Transplanting such cells that are capable of releasing FVIII insitu or into the circulation is an attractive approach for treating clotting factor disorders. Notably, in murine endothelial cell cultures, FVIII production was measured to amount to 0.07IUmillion1 cellsday1. If we extrapolate this to the 81010 liver endothelial cells contained in an adult human liver, the production would cover more than that required by a human adult [15]. Transplanting 2106 mature LSECs, representing 10% of their endothelial compartment, via the portal vein in hemophilia A mice was shown to restore plasma FVIII activity levels to 14%25% of normal, thereby correcting spontaneous bleeding [16].

In addition to endothelial cells, mesenchymal stem cells (MSCs) are also able to secrete FVIII in the cell culture supernatant, which makes them attractive candidates for treating hemophilia. MSCs are increasingly used in several medical areas for repairing organs, such as the heart, bone, cartilage, and liver. These are tissue-resident cells that can be obtained from different tissues and organs, exhibiting the propensity to attach onto surfaces and proliferate invitro. In certain conditions, they differentiate into mature cells, such as bone, cartilage, and adipocyte- or hepatocyte-like cells. These cells express specific surface markers,
such as CD90 and CD105, and are negative for hematopoietic markers like CD45 [17]. However, MSC from different tissues may differ by other characteristics. For example, unmodified MSCs from human liver and lungs produce FVIII in the culture supernatant in much higher quantities compared to human bone marrow-derived MSCs (1.2 and 1.7% vs. 0.12%, respectively, per 105 cells at the 48-h time point) [18].

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