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440 Part V: Therapeutic Principles Chapter 29: Gene Therapy for Hematologic Diseases 441

cell levels were less than 0.4 percent. The fact that both modified HSCs the patient remained with a good quality life with a stable Hgb of 9 to
and T cells failed to repopulate indicated that the in vivo selection of 10 g/dL, transfusion-free, and cancer-free for up to 7 years. The dom-
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modified HIV-resistant cells may be weak or the modified T cells do inant clone might indicate a strong in vivo selection, however, the level
not have a proliferative reaction to HIV infection. In both cases, the of gene modified cells in this patient has been never greater than 21 per-
engraftment rate was very poor (T cell <10 percent and HSC <0.2 per- cent, and the highest level noted in blood was 10.9 percent and in ery-
cent), which could indicate that the modified cells do not have a suffi- throblasts was 3.3 percent, which is below that predicted. The dominant
cient starting number for repopulation. New trials are underway with clone remained stable over time. However, HMGA2 overexpression was
an improved lentiviral vector, engraftment protocols and a methylgua- detected in erythroid cells, which could enhance in vivo selection and
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nine DNA methyltransferase (MGMT) in vivo selection mechanism. proliferation of the corrected cells. Nevertheless, this is but a single case;
The cure for HIV by gene therapy has shown promise, but some clinical whether the in vivo selection of the dominate clone had a significant role
obstacles have to be overcome to achieve success. is not clear. More trials are needed.
DISORDERS OF HEMOGLOBIN HEMOPHILIA
Thalassemia and sickle cell disease represent the most common Hemophilia is an X-linked single-gene defect, of which 70 percent
single-gene defect diseases worldwide (Chaps. 48 and 49). β-Thalas- of affected patients display inheritance and 30 percent develop from
semia is caused by a mutation in the β-globin gene, resulting in reduced de novo somatic mutations (Chap. 124). There are two major forms
adult hemoglobin A (HgbA) and severe anemia. Therefore, gene of hemophilia: hemophilia A, caused by loss-of-function mutations
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therapy is used to express a normal β-globin gene. Many efforts have of the gene encoding clotting factor VIII (FVIII), and hemophilia B,
been made to use stem cell gene therapy. However, success has been the result of mutations in the gene encoding clotting factor IX (FIX).
very limited. Although a weak survival advantage for corrected red Hemophilia A accounts for 80 percent of patients and hemophilia B
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cells at an early mature stage was observed, the in vivo selection alone for 20 percent. The absence of either FVIII or FIX severely impairs
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appears insufficient to achieve a sustained correction. It is predicated the ability to generate thrombin and, subsequently, fibrin, leading to
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that it would require 20 percent of the primitive hematopoietic cells to spontaneous bleeding when the factor levels fall below approximately
be genetically modified, and the gene expressed at near normal levels in 5 percent of normal. Theoretically, gene therapy using a lentiviral vector
those cells, to achieve a definitive therapeutic benefit. Even higher levels that permanently expresses a normal FVIII or FIX gene in the patient
of corrected cells (approximately 100 percent) might be required to cure could cure either disease. However, after 2 decades of intense research,
the disease. The first successful clinical trial was reported in 2007. An gene therapy has been very difficult. FVIII and FIX are produced in
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18-year-old patient with severe β-thalassemia dependent on monthly hepatocytes, not in derivatives of HSCs. Therefore, hemophilia is not
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red cell transfusion since age 3 years, received HSC lentiviral β-globin a circumstance for HSC-based gene therapy. The emerging approach
gene therapy. The viral transduction rate was approximately 30 percent. to gene therapy for hemophilia is by using in vivo gene therapy (see
The patient continued receiving transfusions for 16 months after the Fig. 29–1). In this approach, viral particles are injected into a patient’s
transplantation, at that point the therapeutic HgbA was sufficient and vein, muscle, hepatic artery, or omentum. Initially, five clinical trials
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maintained at a sufficient level until 33 months (Fig. 29–3). During the with retroviral, adenoviral, or AAV vectors failed to achieve long-term
final 21 months, 100 percent of HgbA was from modified cells and the expression of the coagulation factor and no measurable clinical benefit
patient was transfusion-free. However, the therapy effect was later found was observed. However, a trial by a British-American team reported
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to be from a dominant clone (>60 percent of all viral insertion sites in in 2011 showed exceptional results. This group focused on hemophilia
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nucleated blood cells at 24 months), in which the viral insertion causes B. The FIX gene, unlike the FVIII gene, is small and easy to insert into
transcriptional activation of HMGA2 in erythroid cells. Nevertheless, an AAV vector, and 1 to 2 percent of the normal blood levels of FIX
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Monthly transfusion

12 HbA

10
Hemoglobin levels (g/dI) 8 HbA T87Q

6
HbF
4

2
0 HbE
–5 –3 3 4 5 6 9 111213151618192021222425272829303233
Months after trans-gene implant
Figure 29–3. A typical successful gene therapy outcome. In this case a β-thalassemia patient was given a lentiviral vector that contains a HgbA T87Q
transgene. After infusion of modified HSCs 16 months (red arrow), HgbA T87Q (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.)

Kaushansky_chapter 29_p0437-0446.indd 441 9/19/15 12:22 AM