X-LINKED SEVERE COMBINED IMMUNODEFICIENCY
X-SCID is a single-gene-deficient disease caused by mutations in the gene of the interleukin-2 receptor, γIL2RG (Chap. 80).23 Lack of functional γIL2RG results in a lack of T and NK cells and poorly functional B cells. X-SCID is a fatal disease, and without medical interventions, patients often die within the first 2 years of life.23 During 1999 to 2006, 20 X-SCID children were treated in two gene therapy trials.24,25 Because patients lacked an human leukocyte antigen (HLA)-identical donor, precluding stem cell transplantation as a curative therapy, all 20 patients were given a single γ-retroviral vector-mediated gene therapy in which a wild-type γIL2RG gene was delivered into patients’ T cells. From 5 to 12 years of observation after the gene transfer procedure, 17 of the 20 treated participants are alive and display nearly full correction of the T-cell deficiency by genetically modified T cells.24,25
ADENOSINE DEAMINASE DEFICIENCY SEVERE COMBINED IMMUNODEFICIENCY
Adenosine deaminase (ADA) deficiency is an autosomal-recessive inherited disorder caused by mutation of ADA gene on chromosome 20 (Chap. 80). ADA deficiency leads to inhibition of DNA synthesis, which is particularly toxic to lymphocytes because they are some of the most mitotically active cells. This condition ultimately causes SCID. ADA-SCID is almost always fatal by 2 years of age. Gene therapy with a normal ADA gene expressed in autologous HSC is a potentially curative treatment. However, early attempts at gene therapy did not shown any clinical benefit. In the late 1990s, when improved retroviral vector and preinfusion chemotherapy conditioning were instituted, success occurred. Since 2000, 40 patients have been treated in Italy, Great Britain, and the United States.26,27,28 In these studies HSCs were transduced with a γ-retroviral vector encoding the ADA enzyme. Low-intensity conditioning with either busulfan or melphalan was used to increase engraftment of stem cells. In some patients the corrected ADA gene can be detected in blood mononuclear cells for more than 9 years, and the ADA enzyme remains at a normal level.27
Wiskott-Aldrich syndrome (WAS) is the result of a mutation in the gene encoding the WAS protein (WASp).28 The gene is located on the short arm of the X chromosome and WAS is an X-linked recessive genetic disorder. WASP activates actin polymerization in almost all blood cells. Hereditary deficiency in WASP is associated with microthrombocytopenia, recurrent infections, eczema, high incidence of autoimmunity and hematopoietic malignancy (lymphoma or leukemia).29 In 2010, three patients in Italy were administered a lentiviral vector engineered to express WASp following busulfan conditioning. All three WAS patients showed excellent multilineage engraftment with an average of 0.4 to 0.9 correct gene copy per genome persisting to 30 months. Symptoms of WAS improved substantially. Pretreatment eczema resolved between 6 and 12 months after therapy. The frequency and severity of infections progressively decreased, and cytomegalovirus replication was controlled, allowing withdrawal of antiinfectious prophylaxis in two patients. Improvement of platelet count resulted in discontinuation of platelet transfusions.3
LEUKODYSTROPHIES, X-LINKED ADRENOLEUKODYSTROPHY, AND METACHROMATIC LEUKODYSTROPHY
There are two major leukodystrophies, X-ALD and metachromatic leukodystrophy (MLD); both have been successfully treated with gene therapy.4,6 X-ALD is a severe genetic demyelinating disease caused by mutations in the ABCD1 gene on the X chromosome that encodes the adrenoleukodystrophy (ALD) protein, an adenosine triphosphate binding cassette transporter. ALD deficiency leads to the accumulation of very-long-chain fatty acids and progressive demyelination in the central nervous system. In 2009, two French children were reported to have autologous HSC gene therapy with a lentiviral vector encoding wild-type ABCD1.6 The patients were given myeloablative regimen conditioning with cyclophosphamide and busulfan. ALD protein was expressed in 23 and 25 percent of blood mononuclear cells in the two patients. Over 3 years of followup, corrected ABCD1 was found in 7 to 14 percent of granulocytes, monocytes, and T and B lymphocytes. Cerebral demyelination was arrested.6
MLD is an autosomal recessive disorder caused by mutations in ARSA gene in chromosome 22 that encodes arylsulfatase A (ARSA). Deficiency in ARSA leads to sulfatide accumulation, eventually destroying the myelin sheath of the nervous system. In 2013, three children with the disease received autologous HSC gene therapy with a lentiviral vector encoding wild-type ARSA.4 After myeloablative conditioning with busulfan, engraftment was excellent with 45 to 80 percent gene marking levels. ARSA activity was restored to above normal values in the hematopoietic lineages and the cerebrospinal fluid.4 There was a clear therapeutic benefit. In X-ALD and MLD gene therapies, corrected cells did not have an obvious selective advantage. However, in both cases the gene marking levels were high (>10 percent) for as long as 2 years.4,6 These results indicate that successful gene therapy can be achieved with very high viral transduction rates, even absent a transduced cell selection mechanism.
Avoiding Graft-versus-Host Disease
Gene therapy can lead to graft-versus-host disease (GVHD), especially after an allogeneic stem cell transplantation. To prevent GVHD, a suicide gene is introduced in the transferred cells; in the event of GVHD, a prodrug is given to activate the suicide gene to kill the transduced cell. This “safety mechanism” can also guard against uncontrolled proliferation of targeted cells. Traditionally, the suicide gene is herpes simplex virus thymidine kinase (HSV-TK), which can be made “suicidal” in the presence of ganciclovir (GCV). Once GCV is phosphorylated by HSV-TK, it turns to a nucleotide analogue that inhibits DNA synthesis and kills the cell.30 Another new suicide gene system was developed by an inducible caspase-9 protein (iCasp9), which activates the mitochondrial apoptotic pathway. Its use is based on caspase-9 dimerization to an active form. The catalytic domain of caspase-9 is fused to a modified FK506-binding domain that can be homodimerized by a chemical inducer, such as AP1903 (Fig. 29–2). In a clinical trial, four patients who had undergone stem cell transplantation for relapsed acute leukemia were treated with the modified T cells. A single dose of AP1903 was given when GVHD developed, which eliminated more than 90 percent of the modified T cells within 30 minutes and eliminated the clinical signs of GVHD.31 In comparison to HSV-TK, iCasp9 offers several advantages. It induces cell death faster, does not rely on cell proliferation, which makes it ideal for cancer stem cell killing; and, because caspase-9 is downstream of the mitochondria, the sensitivity of cells to its activation should be independent of the antiapoptotic BCL-2 family of proteins, which is often upregulated in hematologic malignancies.
Inducible caspase-9 protein (iCasp9) suicide gene therapy system. A fusion protein contains a mutated FK506 protein linked to the modified caspase 9 (deleted for activation and recruitment domains). The prodrug AP1903 interacts with the FK506 domain and triggers the fusion protein dimerization, which activates caspase-9. Activated caspase-9 initiates the cell caspase cascade (caspases 3 and 7) and induces cell death by apoptosis.
HUMAN IMMUNODEFICIENCY VIRAL INFECTION
The HIV infects helper T cells, such as CD4+ T cells, macrophages, and dendritic cells. The infection kills CD4+ T cells and cripples cell-mediated immunity (Chap. 81). Without treatment, average survival time after infection with HIV is estimated to be 9 to 11 years. In 2007, a gene therapy trial demonstrated that AIDS can be cured. A patient with an HIV infection and acute myeloid leukemia was given allogenic stem cell transplantation from a selected donor, whose two copies of the chemokine (C-C motif) receptor 5 gene (CCR5) were lost because of mutations.32 CCR5 is the major cellular coreceptor used by HIV to infect CD4+ T cells. The patient has since remained off anti-HIV drugs for 7 years. This case generated enormous interest in gene therapy approaches to cure HIV by blocking CCR5 expression. One of the most promising methods is to create loss of function mutations of CCR5 with zinc-finger nucleases (ZFNs). ZFNs are artificial restriction enzymes generated by fusing zinc fingers to a nonspecific double-strand DNA cleavage protein, a truncated Fox1.33 A zinc finger can be engineered to a target 18 to 24 bp sequences in CCR5 DNA. ZFNs can repeatedly cut the DNA at a targeted site and eventually mutate CCR5 when repair errors occur. In a 12-patient clinical trial, the patient’s CD4+ T cells were infected ex vivo with a ZFN-expressing adenoviral vector to disrupt CCR5.34 The modified cells were then reintroduced. CD4 T cells lacking CCR5 are resistant to HIV infection. Ultimately, the CCR5-mutated cells replace those vulnerable CCR5 wild-type cells. The infusion immediately increased the circulating CD4+ T cell count from a median of 488 × 109/L to 1517 × 109/L in the first week. The modified cells were also found in T-cell–rich gut-associated lymphoid tissues. Patients were off antiviral drugs for 84 days during which time the circulating CD4+ T cells dropped while viral load spiked. Antiviral treatment had to be reinstituted.34 The CCR5-mutated CD4+ T-cell count remained stable even during the drug-off period, consistent with their resistance to HIV killing. However, CCR5-mutated CD4+ T cells apparently did not expand quickly, which may explain why in this trial the modified cell infusion alone was insufficient to control the HIV infection. Another trial used a lentiviral anti-HIV small interfering RNA (siRNA) expression vector to modify HSCs.35 Although long-term (18 months) expression of siRNA in multiple blood cell lineages was observed, modified cell levels were less than 0.4 percent. The fact that both modified HSCs and T cells failed to repopulate indicated that the in vivo selection of modified HIV-resistant cells may be weak or the modified T cells do not have a proliferative reaction to HIV infection. In both cases, the engraftment rate was very poor (T cell <10 percent and HSC <0.2 percent), which could indicate that the modified cells do not have a sufficient starting number for repopulation. New trials are underway with an improved lentiviral vector, engraftment protocols and a methylguanine DNA methyltransferase (MGMT) in vivo selection mechanism.36 The cure for HIV by gene therapy has shown promise, but some clinical obstacles have to be overcome to achieve success.
Thalassemia and sickle cell disease represent the most common single-gene defect diseases worldwide (Chaps. 48 and 49). β-Thalassemia is caused by a mutation in the β-globin gene, resulting in reduced adult hemoglobin A (HgbA) and severe anemia.37 Therefore, gene therapy is used to express a normal β-globin gene. Many efforts have been made to use stem cell gene therapy. However, success has been very limited.37 Although a weak survival advantage for corrected red cells at an early mature stage was observed, the in vivo selection alone appears insufficient to achieve a sustained correction.38 It is predicated that it would require 20 percent of the primitive hematopoietic cells to be genetically modified, and the gene expressed at near normal levels in those cells, to achieve a definitive therapeutic benefit. Even higher levels of corrected cells (approximately 100 percent) might be required to cure the disease.38 The first successful clinical trial was reported in 2007.39 An 18-year-old patient with severe β-thalassemia dependent on monthly red cell transfusion since age 3 years, received HSC lentiviral β-globin gene therapy. The viral transduction rate was approximately 30 percent. The patient continued receiving transfusions for 16 months after the transplantation, at that point the therapeutic HgbA was sufficient and maintained at a sufficient level until 33 months (Fig. 29–3). During the final 21 months, 100 percent of HgbA was from modified cells and the patient was transfusion-free. However, the therapy effect was later found to be from a dominant clone (>60 percent of all viral insertion sites in nucleated blood cells at 24 months), in which the viral insertion causes transcriptional activation of HMGA2 in erythroid cells.39 Nevertheless, the patient remained with a good quality life with a stable Hgb of 9 to 10 g/dL, transfusion-free, and cancer-free for up to 7 years.40 The dominant clone might indicate a strong in vivo selection, however, the level of gene modified cells in this patient has been never greater than 21 percent, and the highest level noted in blood was 10.9 percent and in erythroblasts was 3.3 percent, which is below that predicted. The dominant clone remained stable over time. However, HMGA2 overexpression was detected in erythroid cells, which could enhance in vivo selection and proliferation of the corrected cells. Nevertheless, this is but a single case; whether the in vivo selection of the dominate clone had a significant role is not clear. More trials are needed.
A typical successful gene therapy outcome. In this case a β-thalassemia patient was given a lentiviral vector that contains a HgbAT87Q transgene. After infusion of modified HSCs 16 months (red arrow), HgbAT87Q (red) completely replaced HgbA (blue) that was from prior transfusions. At this point, the patient had become transfusion-independent. (Adapted with permission from Cavazzana-Calvo M, Payen E, Negre O, et al: Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia. Nature 16;467(7313):318–322, 2010.)
Hemophilia is an X-linked single-gene defect, of which 70 percent of affected patients display inheritance and 30 percent develop from de novo somatic mutations (Chap. 124). There are two major forms of hemophilia: hemophilia A, caused by loss-of-function mutations of the gene encoding clotting factor VIII (FVIII), and hemophilia B, the result of mutations in the gene encoding clotting factor IX (FIX). Hemophilia A accounts for 80 percent of patients and hemophilia B for 20 percent.41 The absence of either FVIII or FIX severely impairs the ability to generate thrombin and, subsequently, fibrin, leading to spontaneous bleeding when the factor levels fall below approximately 5 percent of normal. Theoretically, gene therapy using a lentiviral vector that permanently expresses a normal FVIII or FIX gene in the patient could cure either disease. However, after 2 decades of intense research, gene therapy has been very difficult. FVIII and FIX are produced in hepatocytes, not in derivatives of HSCs.42 Therefore, hemophilia is not a circumstance for HSC-based gene therapy. The emerging approach to gene therapy for hemophilia is by using in vivo gene therapy (see Fig. 29–1). In this approach, viral particles are injected into a patient’s vein, muscle, hepatic artery, or omentum.43 Initially, five clinical trials with retroviral, adenoviral, or AAV vectors failed to achieve long-term expression of the coagulation factor and no measurable clinical benefit was observed.43 However, a trial by a British-American team reported in 2011 showed exceptional results.15 This group focused on hemophilia B. The FIX gene, unlike the FVIII gene, is small and easy to insert into an AAV vector, and 1 to 2 percent of the normal blood levels of FIX is sufficient to markedly reduce the bleeding risk.43 A new improved AAV vector (AAV8) was used. This vector has a self-complementary genome to improve transduction efficiency, and was designed to produce fivefold higher levels of capsid protein to reduce a potential cytotoxic T-cell response and increase liver tropism. The AAV vector is not genome-integrating and maintains itself as an intracellular episome. Its gene expression in growing cells is transient because episomes may be lost with each cell division. But in quiescent tissues the AAV vector is capable of mediating long-term gene expression as episomal chromatin.44 A single intravenous infusion of the vector was given to six adult male hemophilia B patients who had been treated with recombinant FIX for many years. No notable acute or chronic toxicities were observed. All six patients displayed stable FIX expression at 2 to 11 percent of normal blood levels for 3 years. Four of these patients discontinued recombinant FIX treatment and remained free of spontaneous hemorrhage.15,43,45 The same research team is attempting a similar approach for hemophilia A gene therapy; however, FVIII gene expression has been inefficient. One reason is gene size. The FVIII coding sequence is 7 kb, which far exceeds the normal packaging capacity of AAV vectors. By modifying the B domain, the FVIII size was reduced to a 5.2-kb AAV expression cassette, which is more efficiently packaged. Also, a hybrid liver-specific promoter was introduced into the vector. The resulting new AAV vector has shown high (15 percent of normal) FVIII expression for 20 to 45 weeks in four macaques.46 This AAV vector will be used in a trial of hemophilia A gene therapy in the near future.
Fanconi anemia (FA) is caused by mutations in Fanconi genes, which encode the DNA repair proteins that form a function complex (Chap. 35). FA cells are hypersensitive to DNA crosslinking agents.47 Sixteen Fanconi genes have been described. A defect in one of them will lead to FA. The disease is characterized by a high risk of developing marrow failure and later myelodysplasia, acute leukemia, or cancers of other tissues.47 More than half of patients with FA are the result of FANCA gene mutations; therefore current gene therapy has focused on FANCA insufficiency. Gene therapy for FA is particularly challenging because of the low numbers of HSCs in the stage of marrow failure, and FA cells are extremely sensitive to DNA damage when exposed to myelosuppressive drugs used to condition the patient for stem cell transplantation. In a rare case, two identical twins had inherited FANCA mutations but with normal DNA repair in their blood stem cells. Functional FANCA in blood cells was found to be restored by a spontaneous intrauterine self-correcting somatic mutation in a single HSC. The fact that a single HSC was sufficient to restore a fully normal blood system indicates that FANCA gene therapy may require transduction of only a few HSCs.48
In 2011, an international working group was established to facilitate the development of gene therapy for FA.49 The initial protocol included delivery of a normal FANCA gene by a third-generation lentiviral vector into HSC and increasing HSC number by in vitro HSC expansion using a combination of HOXB4 and DELTA-1 proteins.50
Gene therapy for cancer has been widely exploited. A review was published detailing the new developments in this field.51 This chapter has described a few significant new approaches.
One of the most creative new approaches to cancer-targeted gene therapy is the use of CAR for CLL, in which patient’s T cells were modified to target their own cancers (Chap. 92).7,8 Another strategy is to enhance conventional chemotherapy by protecting the marrow cells through gene transfer. Chemotherapy has a limited therapeutic window because of its severe toxic effect on marrow cells, and its leukemogenic potential. Because the lethal effect of chemotherapy is mainly DNA damage, especially methylating O6-guanine, to overcome the side effects, a strong chemoresistant DNA repair gene, a mutant (P140K) of MGMT was introduced into brain tumor patients’ autologous HSC by a γ-retroviral vector, thus the patient’s transduced marrow progenitor cells could be protected by the modified MGMT, permitting them to tolerate more cycles of chemotherapy. A phase I clinical trial demonstrated that intensification of chemotherapy was feasible, and there was an improvement in therapy outcome and patient survival in these small studies.52,53 A similar result was also observed in a recent clinical trial with a lentiviral vector (reported at the ASH meeting 2014).