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438 Part V: Therapeutic Principles Chapter 29: Gene Therapy for Hematologic Diseases 439

required annealing of the two strands delays gene expression. This lim- Great Britain, and the United States. 26–28 In these studies HSCs were
itation can be overcome by using a self-complementary design in which transduced with a γ-retroviral vector encoding the ADA enzyme.
the two strands of the transgene are on a single hairpin genome in an Low-intensity conditioning with either busulfan or melphalan was used
inverted orientation, which allows quick assembly into a transcription to increase engraftment of stem cells. In some patients the corrected
unit following transduction. 19 ADA gene can be detected in blood mononuclear cells for more than 9
years, and the ADA enzyme remains at a normal level. 27
GENOME-INTEGRATING VIRAL VECTORS
Retroviral Vectors WISKOTT-ALDRICH SYNDROME
A feature of retroviral vectors is that they have the ability to stably Wiskott-Aldrich syndrome (WAS) is the result of a mutation in the gene
incorporate their viral DNA into the host genome, which can result encoding the WAS protein (WASp). The gene is located on the short
28
in long-term expression of the transgene. Most retroviral vectors are arm of the X chromosome and WAS is an X-linked recessive genetic
γ-retroviral or lentiviral vectors. Lentiviral vectors are usually HIV-1 disorder. WASP activates actin polymerization in almost all blood cells.
based, or may also contain elements of simian immunodeficiency virus, Hereditary deficiency in WASP is associated with microthrombocy-
and have at least three advantages over γ-retroviral vectors; first, the len- topenia, recurrent infections, eczema, high incidence of autoimmunity
tiviral vector preintegration complex is able to cross the nuclear mem- and hematopoietic malignancy (lymphoma or leukemia). In 2010,
29
brane in host cells even in the absence of mitosis and, therefore, is able three patients in Italy were administered a lentiviral vector engineered
to transduce nondividing cells, such as HSCs. Second, the lentiviral to express WASp following busulfan conditioning. All three WAS
20
vector preintegration complex is more stable and persists longer, which patients showed excellent multilineage engraftment with an average of
improves the likelihood of integration. Third, γ-retroviral vectors 0.4 to 0.9 correct gene copy per genome persisting to 30 months. Symp-
20
prefer to integrate near gene transcription start sites, such as the CpG toms of WAS improved substantially. Pretreatment eczema resolved
islands and conserved noncoding sequences and conserved transcrip- between 6 and 12 months after therapy. The frequency and severity of
tional factor binding sites, whereas lentiviral vectors are more evenly infections progressively decreased, and cytomegalovirus replication was
distributed, reducing the likelihood of driving the expression of a dele- controlled, allowing withdrawal of antiinfectious prophylaxis in two
terious gene(s). Lentiviral vectors have thus emerged as an important patients. Improvement of platelet count resulted in discontinuation of
22
advance for gene therapy in the hematologic disorders. platelet transfusions. 3
GENE THERAPY FOR HEMATOLOGIC LEUKODYSTROPHIES, X-LINKED

DISEASES ADRENOLEUKODYSTROPHY, AND
X-LINKED SEVERE COMBINED METACHROMATIC LEUKODYSTROPHY
IMMUNODEFICIENCY There are two major leukodystrophies, X-ALD and metachromatic
leukodystrophy (MLD); both have been successfully treated with gene
X-SCID is a single-gene-deficient disease caused by mutations in the therapy. X-ALD is a severe genetic demyelinating disease caused by
4,6
23
gene of the interleukin-2 receptor, γIL2RG (Chap. 80). Lack of func- mutations in the ABCD1 gene on the X chromosome that encodes the
tional γIL2RG results in a lack of T and NK cells and poorly functional adrenoleukodystrophy (ALD) protein, an adenosine triphosphate bind-
B cells. X-SCID is a fatal disease, and without medical interventions, ing cassette transporter. ALD deficiency leads to the accumulation of
patients often die within the first 2 years of life. During 1999 to 2006, very-long-chain fatty acids and progressive demyelination in the cen-
23
20 X-SCID children were treated in two gene therapy trials. 24,25 Because tral nervous system. In 2009, two French children were reported to
patients lacked an human leukocyte antigen (HLA)-identical donor, have autologous HSC gene therapy with a lentiviral vector encoding
precluding stem cell transplantation as a curative therapy, all 20 patients wild-type ABCD1. The patients were given myeloablative regimen
6
were given a single γ-retroviral vector-mediated gene therapy in which conditioning with cyclophosphamide and busulfan. ALD protein was
a wild-type γIL2RG gene was delivered into patients’ T cells. From expressed in 23 and 25 percent of blood mononuclear cells in the two
5 to 12 years of observation after the gene transfer procedure, 17 of the patients. Over 3 years of followup, corrected ABCD1 was found in
20 treated participants are alive and display nearly full correction of the 7 to 14 percent of granulocytes, monocytes, and T and B lymphocytes.
T-cell deficiency by genetically modified T cells. 24,25 Cerebral demyelination was arrested. 6
MLD is an autosomal recessive disorder caused by mutations in
ADENOSINE DEAMINASE DEFICIENCY SEVERE ARSA gene in chromosome 22 that encodes arylsulfatase A (ARSA).
COMBINED IMMUNODEFICIENCY Deficiency in ARSA leads to sulfatide accumulation, eventually destroy-
ing the myelin sheath of the nervous system. In 2013, three children
Adenosine deaminase (ADA) deficiency is an autosomal-recessive with the disease received autologous HSC gene therapy with a lentivi-
4
inherited disorder caused by mutation of ADA gene on chromosome 20 ral vector encoding wild-type ARSA. After myeloablative conditioning
(Chap. 80). ADA deficiency leads to inhibition of DNA synthesis, which with busulfan, engraftment was excellent with 45 to 80 percent gene
is particularly toxic to lymphocytes because they are some of the most marking levels. ARSA activity was restored to above normal values
mitotically active cells. This condition ultimately causes SCID. ADA- in the hematopoietic lineages and the cerebrospinal fluid. There was
4
SCID is almost always fatal by 2 years of age. Gene therapy with a nor- a clear therapeutic benefit. In X-ALD and MLD gene therapies, cor-
mal ADA gene expressed in autologous HSC is a potentially curative rected cells did not have an obvious selective advantage. However, in
treatment. However, early attempts at gene therapy did not shown both cases the gene marking levels were high (>10 percent) for as long
any clinical benefit. In the late 1990s, when improved retroviral vec- as 2 years. These results indicate that successful gene therapy can be
4,6
tor and preinfusion chemotherapy conditioning were instituted, achieved with very high viral transduction rates, even absent a trans-
success occurred. Since 2000, 40 patients have been treated in Italy, duced cell selection mechanism.

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