Page 1748 - Hematology_ Basic Principles and Practice ( PDFDrive )

Chapter 98 Principles of Cell-Based Genetic Therapies 1553

platform for more direct comparison with previous γ-retrovirus and A number of gene therapy trials for ADA deficiency were initiated
lentivirus trials with respect to safety and efficacy. in the early 1990s, targeting retroviral gene transfer into various cell
Strikingly, in contrast to five cases of insertional mutagenesis in types, including peripheral blood lymphocytes, umbilical cord blood,
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the two SCID-X1 trials, no adverse events have been reported in the BM, and CD34 selected stem cells. These early studies failed to
10 patients treated in the ADA-SCID trial, despite a similar RIS produce clear efficacy. By contrast, more recent studies introduced
pattern observed in this patient group. There have been no leukemias key modifications to the gene therapy protocol, including the use of
reported in more than 40 patients treated worldwide (including in a reduced-intensity myelosuppressive conditioning regimen and the
London, Los Angeles, and at the NIH) and most of these have withdrawal of concurrent PEG-ADA replacement. The Milan-based
benefited from gene therapy. The observation of the difference in group of Aiuti and colleagues have reported on their initial experience
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leukemia in these trials has led to the proposal of a “disease effect” with 10 children. Patients were conditioned with 4 mg/kg of
contributing to oncogenesis of γ-chain gene therapy. Woods and busulfan before the infusion of transduced cells. The mean age at the
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colleagues demonstrated lentiviral transduction of γc mice with time of gene therapy was 2.2 years. All children on this trial are
vectors containing the human common γ-chain (cγ) or an inert healthy and thriving, with the longest published follow-up time now
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control gene at very high viral doses. They observed the induction more than 64 months. Gene therapy has resulted in a substantial
of T-cell malignancies in one-third of the animals receiving cγ- increase of lymphocyte counts and normalization of T-cell function.
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transduced cells but not in the control groups. The merit of this very A recent update on this trial, published in abstract form, reports that
limited study was subsequently challenged, primarily on the basis of 18 patients have been treated and are alive and, of these, 15 do not
a viral dose much higher than that used in clinical scenarios and require replacement enzyme treatment.
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incomplete data on the pathogenesis of the malignancies, particularly Similarly, four patients have been treated in London. One
as they relate to the downstream activation level of a key signaling patient that has been reported in detail had been treated with
target of the common γ-chain, Janus kinase 3, in the tumors. In PEG-ADA for 3 years but showed a gradual decline in T-cell numbers
addition, the lentiviral vector used in this study incorporated a hybrid despite effective metabolic correction. Because a matched BM donor
promoter or enhancer element that is extremely powerful and likely was not available, the patient was enrolled on the ADA-SCID gene
to possess a greater transactivating potential than promoter or therapy trial. PEG-ADA replacement was stopped 1 month before
enhancer elements that would be considered for clinical gene therapy gene therapy, and the patient was conditioned with a single dose of
use. More recent occurrence of leukemia in another trial for Wiskott- 140 mg/m2 of melphalan before infusion of the transduced BM
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Aldrich syndrome (WAS; see later) using the same vector backbone CD34 cells. At the time of the last published follow-up, the patient
further challenges the presence of SCID-X1 disease or transgene was 2 years from gene therapy, clinically well, and off prophylactic
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effects in these leukemias. antibiotic therapy. An increase in T-cell numbers and normalization
In summary, thus far, 5 out of 20 patients treated with gene of the proliferative response have been noted. Importantly, no adverse
therapy for SCID-X1 have encountered a life-threatening severe events have occurred thus far in the patients treated for ADA-SCID
adverse event, thought to be triggered by retroviral activation of at these two centers.
LMO2 in 4 patients. Four patients were salvaged with chemotherapy, Aiuti and colleagues recently published a comprehensive genome-
and one patient succumbed to the disease after an unsuccessful wide analysis of RIS of five patients treated in Milan. This paper
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allogeneic BMT. At this point, the use of MLV-based retroviral analyzed the RIS patterns in CD34 cells before infusion as well
vectors with LTR promoter enhancer elements intact is viewed as as RIS in vivo up to 47 months after gene therapy. As anticipated,
contraindicated in this disease by most investigators in the field. The a nonrandom proviral integration pattern, favoring TSS and gene-
continued development of safety-enhanced vectors and the validation dense regions, was observed in the pretransplant cells. RIS observed
of these vectors in clinically relevant systems have emerged as a major in vivo in T cells were additionally enriched for TSS, suggesting the
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priority in the field, and, as noted above, an international trial has occurrence of in vivo selection. More recently, Aiuti and colleagues
recently been reported using a γ-retrovirus that is deleted of LTR have demonstrated that cellular genes in the proximity of the pro-
enhancer elements. Transgene expression is mediated by a weak cel- viral integration site are subject to moderate dysregulation in gene
lular promoter in this vector. Data suggest that early efficacy is modified T-cell clones isolated from patients. However, in contrast
maintained despite lower expression of the cγ-chain and that the to the SCID-X1 trial, no in vivo skewing toward RIS in genes
deletion of the γ-retrovirus enhancer is a key to improved safety. 6 affecting survival, cell cycling, signal transduction, or proliferation
were observed, making a clonal dominance effect appear less likely.
Interestingly, only one RIS was detected at the MDS-EVI1 locus
Adenosine Deaminase Deficiency and became undetectable at later time points. This is in contrast
to the clonal dominance of MDS-EVI1 integration sites observed
ADA is a housekeeping enzyme of the purine metabolic pathway that in the X-linked Chronic Granulomatous Disease trial (see later).
is expressed in all tissues of the body. Deficiency of this enzyme leads Additionally, an overrepresentation of RIS was noted in the proximity
to a buildup of toxic metabolites with detrimental systemic effects, of the CCND2 and LMO2 genes, with a total of 5 out of 523 RIS
including neurodevelopmental deficiencies, sensorineuronal deafness, recovered in vivo. Notably, the CCND2 insertions were detected
and skeletal abnormalities. Importantly, ADA deficiency causes only in the first 2 years of follow-up and not subsequently. LMO2
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abnormal T-, B-, and NK-cell development, resulting in the SCID insertions were also overrepresented in the pretransplant CD34
phenotype. As is the case with the more common SCID-X1, untreated samples, highlighting the fact that the LMO2 gene is a hotspot for
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patients generally succumb to severe opportunistic infections in the retroviral integration in human CD34 cells. The lack of in vivo
first year of life. Treatment strategies used to manage affected patients expansion of clones carrying LMO2 RIS indicates that this integra-
include allogeneic HSCT, enzyme-replacement therapy, and, more tion site may not be sufficient to mediate clonal dominance and
recently, gene therapy. Allogeneic HSCT from a human leukocyte leukemic transformation. Rather, additional cooperating mutations
antigen-matched family donor offers good immunologic and bio- or insertions are required for malignant transformation. The lack of
chemical correction with 73% survival. However, outcomes after malignant transformation in two ADA-SCID trials may point to the
mismatched and haploidentical transplants are less impressive. Like- role of the genetic background or the role of the therapeutic transgene
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wise, the exogenous replacement of ADA, administered in a polyeth- introduced into human CD34 cells. However, the patient cohort
ylene glycol (PEG) conjugate by intramuscular injection on a weekly remains relatively small, and follow-up is still short term. As noted
or twice-weekly schedule, results in systemic detoxification and previously, over 40 patients have now been treated worldwide, with
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immune reconstitution. In the long term, however, about half of the no leukemias reported in any trial in this disease to date. An ongoing
patients receiving PEG-ADA replacement continue to require IVIG new multicenter trial utilizes a lentivirus platform (NCT01279720).
infusions, and some patients show a decline in T-cell numbers over Overall, the genotoxicity profile in the ADA-SCID trials has been
time. sufficiently favorable to continue to recommend this experimental