Page 1750 - Hematology_ Basic Principles and Practice ( PDFDrive )

Chapter 98 Principles of Cell-Based Genetic Therapies 1555

reconstitution in this disease treatment correlated with the dosage of to which inactivation mutations inhibit β-globin expression. However,
gene-corrected cells. other genetic loci can modulate the disease phenotype, for example,
by inducing adult expression of the fetal γ-globin gene, which can be
efficiently incorporated into functional hemoglobin in place of the
Childhood Cerebral X-Linked Adrenoleukodystrophy β-globin chain. In particular, Orkin’s group has recently demonstrated
that the transcription factor BCL11A plays a major role in switching
Childhood cerebral X-linked CCALD is a fatal neurodegenerative γ-globin off and β-globin expression on during the transition from
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disease that results from progressive neural demyelination within the fetal to adult life. Gene therapy of β-thalassemia is complicated by
brain. The defective gene that is responsible for the phenotype, the requirement for an exact stoichiometry of α- and β-globin chains
adenosine triphosphate-binding cassette D1 (ABCD1), encodes a to facilitate efficient assembly of hemoglobin. Thus, an effective gene
transmembrane transport protein that is responsible for the shuttling therapy vector must be able to facilitate high level expression of
of fatty acids into peroxisomes, where they are subsequently degraded. β-globin in the range of that mediated by the normal endogenous
ALD is characterized by an accumulation of very long-chain fatty gene in an erythroid-specific context. Lineage-specific expression of
acids, although the exact pathophysiology of the disease is unknown. the β-globin chain is particularly important in the context of genetic
Cerebral demyelination is associated with inflammation evident on modification of HSC to prevent high-level expression of β-globin in
gadolinium magnetic resonance imaging studies. Most patients pro- nonerythroid hematopoietic lineages.
gress from no symptoms to a vegetative state and death within 8 years The lentiviral vector used in the trial mentioned earlier comprises
of diagnosis. Allogeneic BM transplant has been found to be thera- a SIN configuration with elements from the β-globin locus control
peutic for cerebral demyelination, presumably because of the infiltra- region driving expression of the β-globin cDNA that has been
tion of donor-derived microglia cells into the brain. However, because mutated to enhance β-globin chain stability. Additionally, the vector’s
of the very rapidly progressive nature of the demyelination and the expression cassette is flanked by chromatin insulator elements that
time required to generate mature microglia from transplanted HSCs, function to both prevent silencing of the transgene expression cassette
BM transplant is most effective when administered as soon after the by inhibitory chromatin structure surrounding the integration site
development of demyelination evident by imaging methods as pos- and to prevent the vector-encoded enhancer from modulating expres-
sible. In cases in which CCALD is successfully treated with HSCT, sion of endogenous genes near to the insertion site. An interim report
continued progression of the disease occurs for 18 months to 2 years on the findings of the clinical trial describes that, in 2007, a single
before disease arrest. Myelin inflammation resolves, but lost neuro- patient with severe transfusion-dependent β-thalassemia major
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logic function is not regained in successfully treated patients. Because received CD34 cells transduced with the lentiviral vector described
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of the time constraints imposed upon finding a suitable human earlier. There was a clear demonstration of clinical efficacy because
leukocyte antigen-matched donor and the occurrence of GVHD, the the patient has been transfusion independent for 15 months. Of note,
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genetic correction of autologous BM CD34 cells is an attractive posttransplant molecular analysis revealed a clonal skewing of periph-
experimental therapeutic option. eral blood cells. The clone, which harbors a proviral integration
A phase I gene therapy trial using a recombinant HIV-1–based within the high-mobility group A2 proteins (HMGA2) gene locus, is
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lentiviral vector to deliver the ABCD1 cDNA into CD34 cells has reported to be stable. Insertion of the provirus into this locus resulted
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been reported from Paris. In the three patients who have been in the production of a 3′ truncated mRNA resulting from the intro-
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enrolled to date, a transduction efficiency of 30% to 50% in CD34 duction of a cryptic splice acceptor, present in the vector insulator
cells was achieved, with mean vector copy numbers of 0.6 to 0.7 per element, into intron 3 of the gene. This truncated mRNA, compris-
cell. Patients were preconditioned with cyclophosphamide and ing exons 1–3, was expressed at elevated levels because of a loss of
busulfan, and between 9% and 23% multilineage gene-marked chi- negative posttranscriptional regulation by Let-7 miRNAs because the
merism has been reported in a follow-up period that extends up to miRNA target sequence in exon 5 was lost. HMGA2 has been found
16 months. In the two patients who had been followed for 16 months to be mutated in chromosomal translocations, primarily in benign
after transplant, HSC gene therapy resulted in arrests of neurologic tumors and less often in malignant tumors. Although the clinical
functional loss and resolution of central nervous system inflammation implications of this clonal outgrowth are unclear, this event clearly
that are similar to those achieved with allogeneic transplant. This trial demonstrates that lentiviral vectors can contribute to insertional
is particularly noteworthy in that it is the first trial to report recon- mutagenesis, albeit in this case via modulation of posttranscriptional
stitution of BM cells that have been transduced with a recombinant regulation of gene expression. Indeed, the propensity to insert in
lentiviral vector. Molecular characterization of lentiviral integration coding sequences may lead to an abundance of abnormal mRNA
sites in engrafting cells indicated that reconstitution was polyclonal, splicing variants.
but detailed analysis of genomic loci targeted by this vector has yet Several additional trials are planned using a similar approach to
to be reported. A multisite, biotechnology-sponsored phase II/III express either a sickle-resistant mutated β-globin protein or to
registration trial is currently accruing patients in several countries increase expression of protective fetal hemoglobin, or to reverse the
using a similar lentivirus vector in this disease (NCT01896102). so-called fetal to adult globin switch based on recent data showing
“cure” of humanized sickle mice by genetic deletion of BCL11A
expressing short hairpin RNA against this transcription factor or by
β-Thalassemia gene-edited deletion of the BCL11A gene in HSCs. These trials
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represent important extensions of the technology from very rare
Patients treated in a clinical trial that used a lentiviral vector for the diseases to a disease with a much larger patient population.
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correction of β-thalassemia have recently been reported and subse-
quent updates are in abstract form. 18,19 Two patients treated have
become transfusion independent and a third patient has shown INSERTIONAL MUTAGENESIS
increasing levels of gene-modified red blood cells expressing trans-
genic hemoglobin. Thalassemias result from mutations that attenuate From an early stage in the development of retroviral vectors for gene
the expression of either the α- or β-globin chains that compromise therapy applications, there has been a concern that recombinant
hemoglobin synthesis and thus cause ineffective erythropoiesis. vectors could elicit cellular transformation by altering expression of
Because adult hemoglobin consists of a tetramer of two α- and two either cellular proto-oncogenes or tumor suppressor genes that are
β-chains, inherited mutations at the β-globin locus cause a mismatch proximal to the genomic integration site. This phenomenon, referred
in the ratio of these two chains, thus preventing the correct assembly to as insertional mutagenesis, was characterized as a property of wild-
of the hemoglobin molecule. Clinically, this may result in transfusion- type γ-retroviruses. Having greatly reduced the likelihood that retro-
dependent anemia, which in turn can promote the serious side effect viral gene therapy vectors could generate replication-competent virus,
of iron overload. In general, disease severity correlates with the degree the risk of a recombinant vector being able to transform a cell via