Page 1747 - Hematology_ Basic Principles and Practice ( PDFDrive )

1552 Part IX Cell-Based Therapies

efficiently infect murine and human hematopoietic cells, including of antibodies, including antibody production after immunization,
T lymphocytes, at low multiplicity of infection. 5 were achieved, and the prophylactic administration of IVIG was
discontinued. At almost more than 10 years after gene therapy, these
patients continued to retain a functional immune system, enabling
EXPERIENCE IN HEMATOLOGIC CLINICAL them to live normally.
However, serious adverse events related to gene therapy have been
TRIALS TO DATE reported in four patients in the French trial, occurring 31 to 68
months after gene therapy, and in one patient in the British trial. In
X-Linked Severe Combined Immunodeficiency these patients, untoward effects of viral integration into the genome
resulted in T-cell leukemia, leading to the death of one of the four
Severe combined immunodeficiency (SCID) comprises a number of affected patients. Much research has subsequently been directed at
rare monogenic diseases, with common features including a block in elucidating the mechanism responsible for these adverse events.
T-cell differentiation and impaired B-cell and natural killer (NK) cell Retroviral integration in the proximity of proto-oncogenes, particu-
immunity. Studies of pattern of inheritance, immune function, and larly the LIM domain only 2 (LMO2) promoter, was involved in
genotypes have led to the identification of at least 11 distinct SCID leukemogenesis in three French patients and one British patient. An
conditions. The most common variant of SCID results from the integration of the unaltered γ-chain–encoding viral vector on chro-
deficiency in expression or function of the common cytokine receptor mosome 11q13, near the first exon of the LMO2 gene, led to the
γ chain, which is shared by the receptors for interleukin (IL)-2, IL-4, unregulated transcription of LMO2, giving rise to a T-cell acute
IL-7, IL-9, IL-15, and IL-21. This condition is inherited in a sex- lymphoblastic leukemia (T-ALL)–like lymphoproliferation in the
linked fashion (X-linked SCID or SCID-X1) and accounts for 40% initial two patients. In the patient in the British trial, the integration
to 50% of all SCID cases. SCID-X1 is characterized by abnormal of the vector 35 kb upstream of the LMO2 locus cooperated with
development or function of T, B, and NK cells, although B cells are secondary genetic aberrations, including a gain-of-function mutation
usually present in humans (so-called T-minus, B-plus SCID). Survival of NOTCH1, a deletion at the CDKN2A tumor-suppressor gene
depends on the reconstitution of T-cell development and function by locus, and a translocation of the T-cell receptor β region, to give rise
allogeneic BM transplantation (BMT). If a genotypically matched to T-ALL. LMO2 is a master regulator of human hematopoiesis that
family donor is available, HSCT confers greater than 80% chance of is involved in stem cell growth and is not normally expressed in T
long-term survival. The absence of T and NK cells in the patient cells. However, LMO2 activation has been implicated in some cases
allows for the engraftment of donor cells without preparative chemo- of human T-cell leukemia. In addition, LMO2 transgenic mice have
therapy conditioning; thus, this is the treatment of choice with been shown to develop T-ALL within 10 months. It is increasingly
minimal toxicity. When a genotypically matched family member is clear that retroviral vectors may “turn on” cellular proto-oncogenes
not available, haploidentical donors (e.g., a parent) or closely matched adjacent to their integration site in the genome. The strong promoter
unrelated donors are used, with varying preference center to center, or enhancer activity of the retroviral LTR element shows particular
and a survival rate of 64% to 78% has been reported. These inferior propensity to the upregulation of genes neighboring the integration
outcomes may be attributed to the increased risk of graft rejection or site. Multiple studies now indicate that γ-retroviral vectors, such as
graft-versus-host disease (GVHD), as well as the effects of T-cell the vectors used in the two SCID-X1 trials, preferentially integrate
depletion, immune suppression causing slower immune reconstitu- into the 5′ end of genes near the TSS. In addition, γ-retroviral vectors
tion, or conditioning with increased risk of infection. Haploidentical have been shown to integrate in or near a number of proto-oncogenes
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transplants rigorously depleted of T cells, similar to genotypically that are actively expressed in human CD34 cells. When human
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related transplant, are performed in some institutions without pre- CD34 cells were transduced with retroviral vectors ex vivo, 21% of
parative chemotherapy conditioning; however, B-cell reconstitution retroviral integrations occurred at recurrent insertion sites (“hot
is poor, and the majority of patients require intravenous immuno- spots”), which were highly enriched for proto-oncogenes and growth-
globulin (IVIG) replacement for life. Interestingly, spontaneous controlling genes. A series of papers investigating the vector integra-
partial correction of severe T-cell immunodeficiencies, including tion sites in both SCID-X1 trials and a trial treating adenosine
SCID, has previously been reported, suggesting a selective advantage deaminase (ADA) deficiency, the ADA-SCID trial (see later), observed
of wild-type T cells over defective T cells. a greater-than-random frequency of vector integrations near the TSS
Three independent gene therapy trials aimed at correcting the of genes that are active in HSCs. Interestingly, in the SCID-X1 trial,
immunologic defect of SCID-X1 patients who lack a genotypically a skewing of vector integration site distribution in vivo was noted.
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matched BM donor have been reported. In the initially reported Compared with retroviral integration sites (RIS) recovered from
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7,8
trials, a total of 20 patients have been treated. Despite minor transduced CD34 cells, RIS recovered from T cells in vivo 9 to 30
technical differences in the two protocols, the basic design of both months after transplantation showed an overrepresentation of RIS
gene therapy trials is quite similar: the complete coding region of the within or near genes encoding proteins with kinase activity, transfer-
human γ-chain was cloned into a “first-generation” γ-retroviral vector ase activity, or proteins involved in phosphorous metabolism. This
regulated by the murine leukemia virus (MLV) LTR sequences, which skewing of RIS in vivo suggests a selection of T cells as a result of
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was used to infect BM-derived CD34 cells in vitro. The transduction viral integration in certain growth- and survival-promoting genes. A
occurred in the presence of early acting cytokines (stem cell factor, more recent trial in X-SCID utilizing a SIN γ-retrovirus design
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thrombopoietin, IL-3, and FMS-like tyrosine kinase 3 ligand) and reports early follow-up on nine patients, although the trial is still
the CH296 human fragment of fibronectin described earlier. Cells accruing patients (NCT01129544). The virus design is noteworthy
were subsequently infused without prior conditioning or cytoreduc- in that it is more directly comparable to a lentivirus, since the viral
tive treatment. Minor differences between the two protocols included enhancer elements are deleted and expression of the IL-2 γ-chain is
the uses of a threefold higher concentration of IL-3 and 4% fetal cell from an “internally positioned” cellular promoter. The initial results
serum in the French trial. Additionally, the French investigators used reported efficacy similar to the previous SCID-X1 trials—no leuke-
the amphotropic envelope pseudotype compared with the use of mias and integration analysis that appears safer than those seen in the
gibbon ape leukemia virus envelope in the British trial. Results in previous two trials. In the eight evaluable patients treated in this trial,
both trials have been extremely encouraging. six demonstrated correction of T-cell reconstitution. One patient died
In the French trial, 10 children younger than the age of 1 year prior to full engraftment of gene-modified cells of preexisting viral
were enrolled between 1999 and 2002. Nine out of 10 infants infection that did not resolve after gene therapy. The two failures
developed normal numbers of T and NK cells, with good immune correlated with lower vector copy number in the transduced product
function. In seven of the nine patients who developed T cells, T-cell as a result of inadequate gene transfer. This study is particularly
counts reached normal levels within 3 months and have remained noteworthy as the only human study in which a γ-retrovirus vector
normal at the time of the last published follow-up. Protective levels deleted of enhancer elements has been utilized. It thus provides a