Clinical applications of hematopoietic stem cell gene therapy.
Hematopoietic stem cell transplantation (HSCT) has a half-century history. It is currently an indispensable treatment for not only incurable blood diseases such as aplastic anemia and severe hemolytic anemia, but also malignant hematological diseases such as leukemia and lymphoma. Although allergenic HSCT is also used to treat hereditary diseases, its indications are restricted because of critical complications including regimen-related toxicities involving conditioning, infection, and graft-versus-host disease.
Studies in recent decades have shown that HSCT can have a long-term effect in the treatment of hereditary diseases involving a responsible gene in hematogenous cells. Although the first successful gene therapy using lymphocytes or bone marrow cells for a patient with adenosine deaminase (ADA) deficiency inspired great hope in the future of gene therapy [1-3], subsequent gene therapy using HSCs for patients with X-linked severe combined immunodeficiency (SCID-X1) resulted in tumorigenesis . In addition to the self-renewal and multilineage differentiation capacities of tissue stem cells, HSCs exhibit cell-cycle dormancy, which complicates their use in gene therapy.
However, as technological advances have increased the safety and efficiency of introducing genes into HSCs, gene therapy with HSCs is attracting attention again. In this chapter, advances in the technology of HSC gene therapy, e.g., vector design to avoid genotoxicity and increase transgenic efficiency by taking advantage of the special characteristics of HSCs, are reviewed. In addition, recent studies on HSC gene therapy for various hereditary diseases, such as thalassemia, Fanconi anemia, hemophilia, primary immunodeficiency, mucopolysaccharidosis, Gaucher disease, and X-linked adrenoleukodystrophy (X-ALD) are discussed.
2. Characteristics of HSCs and gene therapy
The concept of the HSC was introduced by Till and McCulloch in 1961 . Although a healthy adult produces approximately 1 trillion blood cells each day, they are considered to originate from a single HSC which can potentially be transplanted into a mouse [6, 7]. Generally stem cells are defined as cells capable of self-renewal and multilineage differentiation. In addition to these two characteristics, HSCs have the capability of cell-cycle dormancy, i.e. to enter a state of dormancy (G0 phase) in the cell cycle and can continue blood cell production over a lifetime while protecting themselves from various kinds of stress .
Fig. 1 shows HSC surface markers and the typical cytokines regulating HSCs. Stem cell factor (SCF) and thrombopoietin (TPO) are important direct cytokine regulators of HSCs. Although SCF promotes the proliferation and differentiation of hematopoietic progenitor cells, it is thought to not be essential for the initiation of hematopoiesis and HSC self-renewal . TPO and its receptor, c-Mpl, are thought to play important roles in early hematopoiesis from HSCs. In contrast to the CD34+CD38-c-Mpl- population, CD34+CD38-c-Mpl+ cells show significantly better HSC engraftment . Mice lacking either TPO or c-Mpl have deficiencies in progenitor cells of multiple hematopoietic lineages . TPO-mediated signal transduction for the self-renewal of HSCs is negatively regulated by the intracellular scaffold protein Lnk [12, 13]. A signal from angiopoietin-1 via Tie2 regulates HSC dormancy by promoting the adhesion of HSCs to osteoblasts in the bone marrow niche and maintains long-term repopulating activity . Although cytokine-induced lipid raft clustering of the HSC membrane is essential for HSC re-entry into the cell cycle, transforming growth factor-β (TGF-β) inhibits lipid raft clustering and induces p57Kip2 expression, leading to HSC dormancy [15, 16]. Recently, the hypoxic niche of HSCs has been demonstrated. It, along with the osteoblastic and vascular niches, are important for HSC dormancy [17-19]. They are targets in HSC gene therapy .
While making a HSC with few opportunities for cell division into a transgenic target, it is important to design a safe and efficient vector for inserting a gene into the host chromosome. Furthermore, since a hematogenous cell also has many cells which exhibit its function in the specialization process to a mature effector cell, it is also important to select differentiation-specific or non-specific promoters or enhancers during the vector design process.
3. Vectors for HSC gene therapy
Vectors derived from the Retroviridae family, RNA viruses with reverse transcriptase activity, are widely used for inserting genes in host chromosomes. Although adeno-associated virus (AAV) vectors can also insert genes into host chromosomes, this process is inefficient and partial. Gammaretroviruses and lentiviruses are members of the Retroviridae family that are commonly used as vectors in HSC gene therapy. Generally, the former is called simply a retroviral vector and the latter is called a lentiviral vector. When a gene is inserted in the chromosome of an HSC with a Retroviridae vector, genotoxicity can occur.
3.1. Gammaretroviral (Retroviral) vectors
Retroviral vectors are commonly constructed from the Moloney murine leukemia virus (MoMLV) genome. Retroviral genomes have a
Expression of a target gene can be inhibited by mechanisms such as methylation of CpG islands in the promoter region, insertion of a negative control region (NCR) into the LTR, and the presence of a repressor binding site (RBS) downstream of the 5′ LTR. Other vectors, such as the murine stem cell virus (MSCV) vector , the myeloproliferative sarcoma virus vector, the negative control region deleted (MND) vector , and the MFG-S vector  were developed to improve the efficiency of transgene expression; they are widely used in clinical applications of gene therapy involving HSCs.
Since the retroviral viral genome cannot cross the nuclear membrane, it can be incorporated into a chromosome only during the phase of mitosis when the nuclear membrane has disassembled. Since many HSCs are thought to exist in a dormant phase, insertions into the HSC genome with a retroviral vector require a proliferation stimulus by cytokines. Although various combinations of cytokines to suppress the decrease in HSC self-renewal have been studied, stem cell factor (SCF), fms-related tyrosine kinase-3 (Flt-3) ligand, interleukin-3 (IL-3), TPO, among others, are commonly used [24, 25].
3.2. Lentiviral vectors
Human immunodeficiency virus type 1 (HIV-1), the representative lentivirus, differs from gammaretroviruses in that it can be incorporated during a non-mitotic phase. This is one advantage of lentiviral vectors in HSC gene therapy.
Both lentiviruses and gammaretroviruses have
Although first-generation lentiviral vectors included modification genes, they were removed in the second generation because it was discovered that the modification genes are not required for infection during non-mitotic phases. In the third generation, further modifications included the deletion of
To improve the gene transfer into HSCs, Verhoeyen and colleagues designed lentiviral vectors displaying “early-acting cytokines” such as TPO and SCF. This vector can promote survival of CD34 positive HSCs and achieve selective transduction of long-term repopulating cells in a humanized mouse model (Fig. 4) [28, 29].
3.3. Genotoxicity of viral vectors
The most serious problem with using viral vectors to incorporate a gene into a chromosome is the potential development of clonal proliferative diseases such as leukemia, which was observed in clinical trials involving gene therapy for SCID-X1 and chronic granulomatous disease (CGD). Although this problem of genotoxicity represents a great hurdle in the development of clinical applications for gene therapy, there is promising ongoing research on the mechanisms underlying genotoxicity and how to avoid it.
The mechanisms of retrovirus-induced oncogenesis are shown in Fig. 5 . In oncogene capture, an acute transforming replication-competent retrovirus captures a cellular proto-oncogene and mediates transformation. This mechanism does not occur in replication-incompetent vectors. Second, the provirus 3′ LTR can trigger increased transcription of a cellular proto-oncogene. Third, enhancers in the provirus LTRs can activate transcription from nearby cellular proto-oncogene promoters. Fourth, a novel isoform can be expressed when transcription from the provirus 5′ LTR creates a novel truncated isoform of a cellular proto-oncogene via splicing. Fifth, an inserted provirus can disrupt transcription by causing premature polyadenylation. The same mechanisms can occur in cellular oncogenesis when a gene is inserted by a retroviral vector .
Even if a gene is inserted into a HSC similarly, it is also known that there are diseases which may develop a tumor, and diseases a tumor is not accepted to be. Each type of virus has a unique integration profile, and the following observations have been made : (a) Different retroviral vectors have distinct integration profiles. (b) The route of entry does not appear to strongly affect distribution of integration sites. VSV-G–pseudotyped HIV vectors have an integration profile similar to HIV virions with the native HIV envelope despite differences in the route of entry. (c) The integration profile is largely independent of the target cell type, although the transcriptional program and epigenetic status of the target cell can influence integration site selection. (d) For lentiviruses, which can integrate independently of mitosis, the cell-cycle status of the target cell has only a modest effect on the distribution of integration sites.
In order to avoid genotoxicity, various SIN vectors have been developed and improved. In general, lentiviral vectors are considered to have a lower risk of oncogenesis than retroviral vectors . However, when a HSC is the target cell, more attention should be required because tumorigenesis can occur when the cell with the inserted gene undergoes differentiation.
4. Clinical applications of HSC gene therapy
Diseases in which gene therapy using HSCs are being studied are shown in Table 1. They are roughly divided into hematological disorders, immunodeficiencies, and metabolic diseases. Most are congenital or hereditary diseases. The characteristic clinical features and recent basic science or clinical studies on HSC gene therapy for each disease are discussed below.
|X-linked severe combined immunodeficiency (SCID-X1)|
Adenosine deaminase deficiency (ADA-SCID)
Chronic granulomatous disease (CGD)
Wiskott-Aldrich syndrome (WAS)
Janus kinase 3 (JAK3) deficiency
Purine nucleoside phosphorylase (PNP) deficiency
Leukocyte adhesion deficiency type 1 (LAD-1)
|Mucopolysaccharidosis (MPS) types I, II, III, VII|
X-linked adrenoleukodystrophy (X-ALD)
Hemoglobin A (HbA), comprising 98% of adult human hemoglobin, is a tetramer with two α-globin and two β-globin chains combined with a heme group. β-thalassemia is an autosomal hemoglobin disorder caused by decreased β-globin chain synthesis. Although individuals with β-thalassemia minor (heterozygote) may be asymptomatic or have mild to moderate microcytic anemia, β-thalassemia major (homozygote) progresses to serious anemia by one or two years of age, and hemosiderosis, iron overload caused by transfusion or increased iron absorption, develops. Since most patients develop life-threatening complications such as heart failure by adolescence, HSCT has been performed in patients with advanced disease . In recent years, gene therapy using a lentiviral vector containing a functional β-globin gene has been performed in an HbE/ β-thalassemia (βE/ β0) transfusion-dependent adult male, who subsequently did not require transfusions for over 21 months .
The human β-globin locus is located in a large 70kb area which also contains some β-like globulin genes (ε, Gγ, Aγ, δ, β). Gene switching takes place according to the development stage, and the β-globin gene is transcribed and expressed specifically after birth. A powerful enhancer called the LCR (locus control region) exists on the 5′ side of the promoter. The LCR contains five DNase I hypersensitive sites, referred to as HS5 to HS1 starting from the 5′ side. Furthermore, HS5 contains CCCTC-binding factor (CTCF)-dependent insulator.
The structure of the lentiviral SIN vector used in gene therapy for β-thalassemia is shown in Fig. 6. To improve safety, two stop codons were inserted into the packaging signal (ψ) of GAG, the HS5 portion with insulator activity was deleted, and two copies of the 250 base pair (bp) core of the cHS4 chromatin insulators (chicken β-globin insulators) were inserted in the U3 region of the HIV 3′ LTR. Furthermore, the amino acid at the 87th position of β-globin was changed from threonine to glutamine. This altered β-globin can be distinguished from normal adult β-globin by high performance liquid chromatography (HPLC) analysis in individuals receiving red blood cell transfusion and β+-thalassemia patients .
A clinical study using this vector was performed in two β-thalassemia patients. As with autologous bone marrow transplantation, some of the patients’ marrow cells were cryopreserved as a backup. The lentiviral vector particles containing a functional β-globin were introduced into the remaining cells. After the transfected cells were cultured for one week
The first patient failed to engraft because the HSCs had been compromised by how they were handled, not because of any issues with the gene therapy vector, and ultimately used backup bone marrow. The second patient, as described previously, achieved long-term β-globin production; one-third of the patient’s hemoglobin was produced by the genetically modified cells .
Furthermore, the detailed examination of the transgenic cells showed significantly increased expression of high mobility group AT-hook 2 (HMGA2), which interacts with transcription factors to regulate gene expression, in the clones where gene insertion occurred in the
Recently, researchers generated a LCR-free SIN lentiviral vector that combines two hereditary persistence of fetal hemoglobin (HPFH)-activating elements, resulting in therapeutic levels of Aγ-globin protein produced by erythroid progenitors derived from thalassemic HSCs . Both lentiviral-mediated γ-globin gene addition and genetic reactivation of endogenous γ-globin genes are considered potentially capable of providing therapeutic levels of hemoglobin F to patients with β-globin deficiency . In addition, a trial of γ-globin induction with β-globin production using mithramycin, an inducer of γ-globin expression, to remove excess α-globin proteins in β-thalassemic erythroid progenitor cells was reported .
4.2. Fanconi anemia
Fanconi anemia is a hereditary disease characterized by cellular hypersensitivity to DNA crosslinking agents. It leads to bone marrow failure, such as aplastic anemia, by approximately eight years of age. Since there is a high risk of developing malignancy, HSCT has been performed as a curative treatment for bone marrow insufficiency. Although the ten-year probability of survival after transplant from an Human leukocyte antigen (HLA) -identical donor is over 80%, results with other donors are not satisfactory. HSC gene therapy is considered an alternative in cases where there is no HLA-identical donor available [38-40].
There are currently 13 discovered Fanconi anemia complement groups and 13 distinct genes (
Engraftment efficiency of
In recent years, the design of lentiviral vectors used for gene therapy in Fanconi anemia has improved. Although the vav and phosphoglycerate kinase (PGK) promoters are relatively weak, physiological levels of
There was a recent interesting report on the use of induced pluripotent stem cells (iPS cell). Instead of introducing a repaired gene into the HSCs of a patient with a
Hemophilia is a common congenital coagulopathy caused by coagulation factor VIII (hemophilia A) or IX (hemophilia B) deficiency. Although the genes encoding both factor VIII (Xq28) and factor IX (Xq27) are located on the X chromosome and most cases are X-linked, many sporadic variations have been reported. Factor substitution therapies have been used to treat hemophilia for many years. However, there is great hope for gene therapy with hemophilia because coagulation factors have short half-lives (factor VIII, 8 to 12 hours; factor IX, 18 to 24 hours), and an inhibitor is produced in many cases. Furthermore, it is possible for gene therapy to suppress immunogenicity by introducing a mutant protein that lacks the domain with which the inhibitor interacts. Since both coagulation factors are usually produced in the liver, there are few studies involving HSCs. In addition to hepatocytes, trials introducing the modified gene directly into splenic cells, endothelial cells, myoblasts, fibroblasts, etc. have been reported [50-52]. Since the factor IX gene (34 kb) is smaller than the factor VIII gene (186 kb), hemophilia B gene therapy can be possible with an adenovirus vector or an AAV vector. Therefore, hemophilia B is progressing more as a field of gene therapy research even through there are five times more patients with hemophilia A [51-53].
Recently, human factor VIII variant genes were successfully introduced into the HSCs of a mouse with hemophilia A resulting in therapeutic levels of factor VIII variant protein expression. This variant factor VIII has changes in the B and A2 domain in addition to the A1 domain for improved secretion and reduced immunogenicity (wild-type factor VIII has six domains, A1, A2, B, A3, C1, and C2) . To ameliorate the symptoms of hemophilia A, partial replacement of the mutated liver cells by healthy cells in hemophilia A mice was challenged with allogeneic bone marrow progenitor cell transplantation. In this study, the bone marrow progenitor cell-derived hepatocytes and sinusoidal endothelial cells synthesized factor VIII, showing that autologous gene-modified bone marrow progenitor cells have the potential to treat hemophilia .
4.4. Primary immunodeficiencies
Although HSCT has been widely performed as curative treatment for primary immunodeficiencies, gene therapy has been considered when there is no HLA-identical donor available. As previously shown, the first successful gene therapy was performed in a patient with ADA deficiency in the U.S. in 1990. Since the gene was introduced into T lymphocytes, frequent treatment was required. However, this treatment was associated with an unacceptable level of toxicity. Since transfected vector and normal ADA gene expression in T lymphocytes continued for two years after the cessation of treatment , gene therapy attracted attention. With advances in HSC gene-transfer technology, gene therapy for many primary immunodeficiencies can now be considered .
SCID-X1 is an X-linked disease caused by deficiency of the common γ (γc) chain in the IL-2 receptor. Because the γc chain is common to the IL-4, IL-7, IL-9, IL-15, and IL-21 receptors, in SCID-X1 patients, there are defects in T and natural killer (NK) cells, and B cell dysfunction are usually observed . Patients begin suffering from various infections starting several weeks after birth. Without curative treatment, such as HSCT, patients die in infancy.
In SCID-X1, since T cells are lacking, engraftment of the gene-transduced cells can be achieved without pre-conditioning therapy. In the clinical studies of SCID-X1 patients in France and the U.K., the MFG retroviral vector was used with HSCs obtained from the patient. After gene therapy, many patients had improvements in immune function. However, since the genes regulating lymphocyte proliferation, such as
Although continuous T cell production was founded in many cases, there was little reconstruction of myeloid cells and B cells, and some patients required continuous immunoglobulin substitution therapy. The use of conditioning therapy is also related to immunological reconstruction after γc chain gene therapy. There is decreased NK cell reconstruction without conditioning therapy, so conditioning chemotherapy is required for the engraftment of undifferentiated stem cells . A trial of SCID-X1 gene therapy in the U.S. involved three patients ranging from 10 to 14 years of age. They had poor immunological recovery after allergenic HSCT and T cell recovery was only observed in the youngest patient, suggesting there is a limit to the recovery of the function of the thymus in older children .
To study whether activation of genes near the region of gene insertion or inserted γc chain gene expression itself induces oncogenicity during SCID-X1 gene therapy, a study of the human γc chain gene being expressed under the control of the human CD2 promoter and LTR (CD2- γc chain gene) was performed in mice. When the CD2- γc chain gene was expressed in transgenic mice, a few abnormalities involving T cells were observed, but tumorigenesis was not observed and T and B cell functions were recovered in γc chain-gene deficient mice. This study demonstrated that when the γc c chain gene is expressed externally, SCID-X1 may be treated safely .
Although SIN vectors were developed from earlier retroviral  or lentiviral vectors  to reduce the risk of oncogenicity in SCID-X1 gene therapy, genotoxicity unrelated to mutations in gene insertion regions or γc chain gene overexpression have been reported with lentiviral vectors in recent years, and it seems that more sophisticated vector development is required .
ADA is an enzyme that catalyzes the conversion of purine metabolism products adenosine and deoxyadenosine into inosine or deoxyinosine. ADA-SCID is an autosomal recessive disease that results in the accumulation of adenosine, deoxyadenosine, and deoxyadenosinetriphosphate (dATP). Accumulated phosphorylated purine metabolism products act on the thymus and cause the maturational or functional disorder of lymphocytes. Because ADA-SCID patients have both T and B cell production fail, patients have a severe combined immunodeficiency disease with a clinical presentation similar to SCID-X1 results, but unlike SCID-X1, many patients have a low level of T cells. Although enzyme replacement therapy with polyethylene glycol–modified bovine ADA (PEG-ADA) was developed to treat ADA-SCID, it is limited by the development of neutralizing antibodies and the cost of lifelong treatment.
In ADA-SCID, since T cell counts are increased by PEG-ADA, gene therapy to increase peripheral T cell counts was attempted during the early stages of gene therapy. Although adverse events were not observed and continuous expression of ADA was achieved in many patients, reconstruction of immune function was not obtained and substitution therapy with PEG-ADA remained necessary. Therefore, HSCs were no longer the target of gene therapy for ADA-SCID. Since ADA-SCID patients have T cells, nonmyeloablative conditioning was performed to achieve gene-transduced HSC engraftment [25, 65].
In a joint Italian-Israeli study started in 2000, ten ADA-SCID children were infused with CD34 positive cells transduced with a MoMLV retroviral vector containing the
As with SCID-X1, the retroviral vector gene insertion region is also near genes that control cell proliferation or self-duplication, such as
CGD is a disease caused by an abnormality in nicotinamide dinucleotide phosphate (NADPH) oxidase expressed in phagocytes, resulting in failure to produce reactive oxygen species and decreased ability to kill bacteria or fungi after phagocytosis. NADPH oxidase consists of gp91phox (Nox2) and p22 phox which together constitute the membrane-spanning component flavocytochrome b558 (CYBB), and the cytosolic components p47phox, p67phox, p40phox, and Rac. CGD is caused by a functional abnormality in any of these components. Mutations in
In the initial trials of CGD gene therapy without any conditioning therapy,
In a German study where
Recently, next-generation gene therapy for CGD using lineage- and stage-restricted lentiviral vectors to avoid tumorigenesis  and novel approaches involving iPSs derived from CGD patients using zinc finger nuclease (ZFN)-mediated gene targeting were studied . Specific gene targeting can be performed in human iPSs using ZFNs to induce sequence-specific double-strand DNA breaks that enhance site-specific homologous recombination. A single-copy of
4.4.4. Wiskott-Aldrich Syndrome (WAS)
WAS is a severe X-linked immunodeficiency caused by mutations in the gene encoding the WAS protein (WASP), a key regulator of signaling and cytoskeletal reorganization in hematopoietic cells. Mutations in
In one preclinical study introducing the
SIN lentiviral vectors using the minimal domain of the WAS promoter or other ubiquitous promoters, such as the PGK promoter, are currently being developed for WAS gene therapy. Preclinical studies using the HSCs obtained from mice or human patients have yield good results in terms of gene expression and genotoxicity [86-90].
Since a study using human embryonic stem cells (hESCs) and WAS-promoter–driven lentiviral vectors labeled by green fluorescent protein (GFP) showed highly specific gene expression in hESCs-derived HSCs, the WAS promoter will be used specifically in the generation of hESC-derived HSCs .
4.4.5. Janus Kinase 3 (JAK3) deficiency
JAK3 deficiency is characterized by the absence of T and NK cells and impaired function of B cells, similar to SCID-X1. Treatment consists of HSCT with an HLA-identical or HLA-haplo-identical donor, often the parents of the patient, with T cell depletion. Engraftment is successful in most cases.
Although the recovery of T cell function is usually observed after HSCT, there are usually no improvements in B or NK cell function . One case report involved introduction of
4.4.6. Purine Nucleoside Phosphorylase (PNP) deficiency
PNP metabolizes adenosine into adenine, inosine into hypoxanthine, and guanosine into guanine. PNP deficiency is an autosomal recessive metabolic disorder characterized by lethal T cell defects resulting from the accumulation of products from purine metabolism.
In PNP-deficient mice, transplantation of bone marrow cells transduced with a lentiviral vector containing human
4.4.7. Leukocyte Adhesion Deficiency type 1 (LAD-1)
LAD-1 is a primary immunodeficiency disease caused by abnormalities in the leukocyte integrin CD11/CD18 heterodimer due to mutations in the
In order to suppress gene activation near the gene insertion region in CLAD and to obtain the sufficient expression of the
4.5. Mucopolysaccharidosis (MPS)
MPS is a general term for diseases characterized by glycosaminoglycan (GAG) accumulation into lysosomes as a result of deficiencies in lysosomal enzymes that degrade GAG. Although there are more than ten enzymes that are known to degrade GAG, MPS is divided into seven types: type I (α-L-iduronidase deficiency, Hurler syndrome, Sheie syndrome, Hurler-Sheie syndrome), type II (iduronate sulfatase deficiency, Hunter syndrome), type III (heparan N-sulfatase deficiency, α-N-acetylglucosaminidase deficiency, α-glucosaminidase acetyltransferase deficiency, N-acetylglucosamine 6-sulfatase deficiency, Sanfilippo syndrome), type IV (galactose 6-sulfatase deficiency, Morquio syndrome), type VI (N-acetylgalactosamine 4-sulfatase deficiency, Maroteaux-Lamy syndrome), type VII (β-glucuronidase deficiency, Sly syndrome), and type IX (hyaluronidase deficiency). Type II is X-linked; the other types are autosomal recessive. Although lysosomes are found in almost all cells, MPS mainly affects internal organs such as the brain, heart, bones, joints, eyes, liver, and spleen. The extent of disease, including mental retardation, varies with MPS type.
In types I, II, and VI, enzyme replacement therapy is performed. HSCT is performed in types I, II, IV, and VII. Gene therapy for types I, II, III, and VII type have been investigated. There are trials using an AAV or adenovirus vector to insert the modified gene into various cell types, including hepatocytes, muscle cells, myoblasts, and fibroblasts .
The first study of HSC gene therapy for MPS using a retroviral vector was performed on type VII mice in 1992, resulting in decreased accumulation of GAG in the liver and spleen but not in the brain and eyes . Subsequent studies in type I and III animal models showed decreases in GAG accumulation in the kidneys and brain. Introductory efficiency and immunological reactions are considered challenges in HSC gene therapy for MPS .
Restoring or preserving central nervous system (CNS) function is one of the major challenges in the treatment of MPS. Since replaced enzymes easily cannot pass the blood-brain barrier (BBB), a high dose of enzyme is needed to improve CNS function. Gene therapy faces the same challenge. Even with high expression of enzyme by, for example, hepatocytes, the BBB prevents efficient delivery into the CNS. When a lentiviral vector is directly injected into the body, gene expression in brain tissue is observed, although the underlying mechanism is unknown. There are also trials where AAV vectors are directly injected into the CNS of mice or dogs and gene expression was observed in brain tissue .
Recently, a lentiviral vector using an ankyrin-1-based erythroid-specific hybrid promoter/enhancer (IHK) was used with HSCs to obtain gene expression only in erythroblasts for type I MPS. This approach resulted in decreased accumulation of GAG in the liver, spleen, heart, and CNS via enzyme expression in erythroblasts .
4.6. Gaucher disease
Gaucher disease is the most common lysosomal storage disorder. It is caused by deficiency of glucocerebroside-cleaving enzyme (β-glucocerebrosidase), resulting in the accumulation of glucocerebroside in the reticuloendothelial system . This autosomal recessive disease presents with hepatosplenomegaly, anemia, thrombocytopenia, and convulsions with or without mental retardation. It is classified into three types based on the clinical course or existence of neurological symptoms: type I (non-neuropathic, adult type), type II (acute neuropathic, infantile type), and type III (chronic neuropathic, juvenile type). Enzyme replacement therapy has been established in type I. As with MPS, since it is difficult to improve CNS symptoms with enzyme replacement therapy, HSCT is used, especially with type III. Gene therapy is considered in cases with little improvement with enzyme replacement therapy .
For Gaucher disease without CNS symptoms, a animal model using an AAV vector to produce enzyme in hepatocytes yielded good results . HSC gene therapy using a retroviral vector was attempted in type I mice. The treated cells had higher β-glucocerebrosidase activity than the HSCs from wild-type mice. Glucocerebroside levels normalized five to six months after treatment and no infiltration of Gaucher cells could be observed in the bone marrow, spleen, and liver . In recent years, development of lentiviral vectors including the human glucocerebrosidase gene  and low-risk HSCT with nonmyeloablative doses of busulfan (25mg/kg) and no radiation therapy have been attempted in mice .
X-ALD is a peroxisomal disease in which a lipid metabolism abnormality causes demyelination of CNS tissues and dysfunction of the adrenal gland. It results from mutations in the ATP-binding cassette sub-family D (
One study has reported the introduction of wild-type
Gene therapy using HSCs was outlined. HSCT with HSCs can replace all of the patient’s original HSCs with donor HSCs. Therefore, gene therapy using HSCs is an alternative if the patient does not have an HLA-identical donor or cannot tolerate the conditioning regimen or other HSCT-related side effects. Fully myeloablative or nonmyeloablative conditioning regimens are still necessary to eliminate potential competition within the bone marrow compartment, in an attempt to increase the number of gene-modified HSCs or progenitors that produce the therapeutic enzyme or protein. With gene therapy, eliminating the risk of immune reactions against the transgene is necessary. Lentiviral vectors in clinical use must not be contaminated by replication-competent recombinant vectors related to the parent HIV-1 virus. The main risk of retrovirus- or lentivirus-mediated gene therapy may prove to be insertional mutagenesis caused by random retroviral integration leading to activation of proto-oncogenes or inactivation of tumor-suppressor genes, ultimately leading to malignancy . However, with advances in gene introduction technology, such as the development of the SIN vector and advances in cell or gene-region targeting, gene therapy can be done more safely and efficiently. Furthermore, since cells more immature than HSCs, i.e., iPS cells, are available, further advances in HSC gene therapy are expected in the future.