Although there have been some clinical successes using genetically engineered HSCs, additional diseases are candidates for HSC-based gene therapy interventions. For example, hemophilia A is an ideal target for gene therapy, and recent preclinical studies indicate that the major hurdles impeding a successful HSC-based hemophilia A therapy have been overcome. The benefits/risk ratios for treating hemophilia with gene therapy has been discussed extensively, and the consensus is that gene therapy will be the future treatment for hemophilia [45
]. Current therapies for hemophilia are hampered by the high cost of treatment, the demanding schedule required to obtain maximal prophylactic benefit, the potential of inhibitor formation, and arthropathy due to joint bleeding, which can occur under ideal treatment conditions. The high cost of hemophilia A treatments result in only approximately 25% of the world hemophilia A population being treated, which can be considered the number one problem with current treatment options. HSC-based gene therapy can provide a one-time treatment resulting in a lifetime cure for this disease.
Hemophilia A is not treatable by hematopoietic stem cell transplantation alone due to the fact that hematopoietic cells are not an endogenous source of coagulation factor VIII (fVIII). However, hemophilia A is amenable to treatment using HSC transplantation (HSCT) gene therapy. Hemophilia A results from deficiency of plasma fVIII activity. Since fVIII is a secreted protein that functions in the bloodstream at sites of vascular injury, it theoretically can be produced by any cell type with access to the bloodstream. Genetic manipulation of a single HSC facilitates the generation of millions of progeny cells, which if an integrating virus was used to perform the gene transfer also will contain the genetic modification of interest, e.g. a functional fVIII transgene. Therefore, HSCs are an ideal drug delivery vehicle for the treatment of hemophilia A. In the current section, we describe advances in HSCT gene therapy for hemophilia A using it as a model disease and example for the use of HSCs as vehicles for therapeutic nucleic acid and protein delivery.
The first hemophilia A preclinical HSCT gene therapy study was conducted by Evans and Morgan in the 1990’s [46
]. A murine leukemia virus-based γ-retroviral vector was used to deliver a human fVIII transgene to murine bone marrow cells, which subsequently were transplanted into recipient hemophilia A mice that were conditioned with a lethal dose of total body irradiation (TBI). Correction of the fVIII deficiency was not achieved in this initial study presumably due to low level biosynthesis of fVIII from hematopoietic cells. Subsequently, it was shown by Tonn and colleagues that, using in vitro
models, genetically-modified erythroid and/or megakaryocytic cell lines secreted higher levels of fVIII than did lymphoblastoid or T-cell leukemia cell lines suggesting a cell lineage-specific biosynthesis differential. This study instilled renewed enthusiasm for HSCT gene therapy and in 2004 and 2005, Hawley and colleagues reported the first preclinical studies successfully utilizing HSCT gene therapy to achieve correction of the fVIII deficiency in hemophilia A mice to a predicted therapeutic level (≥ 5% of the normal human level or 0.05 units/ml fVIII activity). In their study, they used a bicistronic γ-retroviral vector encoding an EGFP reporter gene in addition to the human fVIII transgene. This modification allowed for the selection of genetically-modified cells by fluorescence activated cell sorting prior to transplantation in order to maximize the engraftment of genetically modified cells and thus increase fVIII production levels [47
]. Although this technique was successful in the mouse model, it is not clinically feasible and further advancements were needed. To improve upon fVIII expression levels, they incorporated modifications to the human fVIII transgene itself that had been shown by others to increase the secretion rate of human fVIII [49
]. Incremental improvement also was made by integrating both an enhanced fVIII transgene containing amino acid residues designed for improved expression and decreased immunogenicity as well as a safety optimized retroviral vector gene delivery system [51
Another successful approach, adopted by our group, was the utilization of the high-level expression property of the orthologous porcine fVIII molecule. The high expression property was identified during the development of a recombinant porcine fVIII product designed, and currently in clinical trials, for the treatment of both congenital and acquired hemophilia A patients with anti-fVIII inhibitory antibodies [52
]. We have shown in a series of studies that the high expression property of porcine fVIII functions effectively in a HSCT gene therapy setting. In the first study, recombinant murine stem cell virus-based vector encoding a porcine fVIII transgene was used to genetically-modify murine HSCs, which were transplanted into hemophilia A mice preconditioned with TBI [55
]. All mice demonstrated complete (>100% of the normal human plasma fVIII activity level) and sustained correction of circulating fVIII activity levels. Life-long restoration of fVIII activity suggested that genetically-modified HSCs engrafted, proliferated, and expressed fVIII at levels significantly greater than that achieved by Hawley’s group using human fVIII variants. This original study demonstrated proof-of-concept that porcine fVIII high-expression elements function effectively in vivo
in a gene therapy setting. Further progress in the field of HSCT gene therapy for hemophilia A focused on improving the safety profile. For example, in the original studies by Hawley and ourselves, TBI was used as a marrow conditioning regimen. Due to the significant risk of long-term side effects and short-term mortality, TBI is not appropriate for the treatment of patients with hemophilia A especially those that have access to fVIII replacement products. Therefore, investigation into the use of more clinically-relevant HSCT regimens that maintain efficient engraftment and fVIII expression while limiting the development of anti-fVIII inhibitors was necessary. Therefore, we subsequently tested several reduced intensity conditioning regimens and identified several that performed successfully including costimulation blockade (anti-CD40L and CTLA4-Ig) and a combination of busulfan and anti-thymocyte serum (ATS) [56
]. Under these conditioning regimens, no mice developed inhibitory anti-fVIII antibodies or obvious leukemic transformations. One conclusion drawn was that T-cell-specific suppression/cytoreduction was a critical component to the successful engraftment of genetically-modified HSCs encoding fVIII. In a related study, we demonstrated that HSCT gene therapy incorporating the high expression porcine fVIII transgene could correct the fVIII deficiency and eradicate pre-existing anti-human-fVIII inhibitors [57
]. This was the first report showing successful treatment of hemophilia A using nucleic acid therapy in the context of fVIII inhibitors and the only report demonstrating inhibitor elimination. As hemophilia A patients with fVIII inhibitors are the most challenging and costly population to treat clinically, they represent an ideal patient population for future gene therapy clinical trials.
The observation that correction of fVIII activity levels can be achieved by a small percentage of genetically-modified hematopoietic cells harboring low proviral copy number supports the feasibility of successfully translating this strategy to human patient care. Clearly, high expression porcine fVIII sequence elements overcome the low-expression barrier observed using standard human fVIII constructs and, more recently, we demonstrated that a high expression hybrid human/porcine (HP)-fVIII construct with as little as 10% porcine amino acid sequence functions indistinguishably in a HSCT gene therapy [58
]. In this study, recombinant lentiviral vectors encoding HP-fVIII were used to genetically-modify human hematopoietic cell lines and murine HSCs, the latter of which were transplanted into hemophilia A mice. Similar to the results obtained using the porcine fVIII transgene, HP-fVIII demonstrated significantly greater expression levels than BDD human fVIII supporting its continued development towards clinical application.
One complexity of expressing a heterologous protein in hematopoietic cells is the diversity of this cell population. HSCs give rise to the three committed cell lineages, erthroid, myeloid and lymphoid cell types, each of which contains several distinct subpopulations. For example, myeloid progenitor cells give rise to monocytes, macrophages, neutrophils, eosinophils and basophils. Each differentiated cell type is presumed to possess differential protein synthesis capacity and specificity. These cellular characteristics are important considerations when determining the appropriateness of HSCT gene therapy for a specific disease. For example, it is established that over-expression of fVIII induces endoplasmic reticulum stress in mammalian cells through the unfolded protein response pathway [59
]. Prolonged ER stress can lead to apoptosis. Therefore, a relevant concern in HSCT gene therapy for hemophilia A is cellular toxicity in hematopoietic stem, progenitor and terminally differentiated cell lineages due to fVIII expression. Research in this area is ongoing, but in preliminary studies, there have been no reports of toxicity due to fVIII expression following HSCT gene therapy.
If lineage-specific toxicity was observed, one possible solution would be directed expression into lineages that are not sensitive to transgene-related toxicity. In addition, lineage specific expression can limit enhancer-related insertional mutagenesis issues because promoters for lineage expression are only active in a limited subset of cells. Along this line of pursuit, two laboratories have reported the specific targeting of fVIII transgene expression to the megakaryocyte/platelet hematopoietic lineage [60
]. In concept, the fVIII transgene is transcribed and fVIII biosynthesis occurs in megakaryocytes and the resulting translation product is stored in platelet granules until release at the time of platelet activation. For these studies, DNA constructs encoding a BDD human fVIII transgene under the control of various integrin promoters were used to generate transgenic mice or incorporated into self-inactivating lentiviral vectors and delivered through HSCT gene therapy to recipient hemophilia A mice. These studies provided proof-of-concept that platelet-directed fVIII expression could be used to correct the bleeding phenotype in hemophilia A mice as assessed by tail-clip bleeding assay despite the lack of circulating fVIII activity. Another facet of this strategy is that fVIII stored in platelets is protected from fVIII inhibitors and retains in vivo
efficacy even in the presence of high titer inhibitors. However, recent results obtained by Poncz and colleagues dispute the interpretation of efficacy using the tail-clip bleeding assay. They conclude that the tail-clip assay is overtly-sensitive to small amounts of fVIII activity due to the nature of the hemostatic challenge and relevant murine physiology [64
]. Despite significant progress, further preclinical and clinical testing will be necessary to determine the translatability of this unique directed expression approach to HSCT gene therapy for hemophilia A. Other hematopoietic cell populations of interest for targeted fVIII expression include plasma B cells, due to their adaptation to the biosynthesis of large quantities of high molecular weight glycoproteins (i.e. immunoglobulins), and erythrocytes, due to their abundant nature and lack of oncogenic disposition.
As a model disease for HSCT gene therapy, hemophilia A possesses both desired and disruptive characteristics. However, much research has been invested in this therapeutic arena and significant progress has been made. Although there has not yet been a clinical trial for HSCT gene therapy of hemophilia A, it likely is a not too distant reality based on recent advancements in the field of HSCT gene therapy for other monogenic disorders and the desire for a cure to this physically and economically debilitating disease. Overall, when the potential benefits and risks of gene therapy of hemophilia are weighed with those associated with conventional therapy, the consensus among key opinion leaders in hemophilia is that gene therapy represents the future of hemophilia care [45