Page 1749 - Hematology_ Basic Principles and Practice ( PDFDrive )

1554 Part IX Cell-Based Therapies

therapy to patients and families lacking a perfectly matched sibling in vitro and failed to engraft in immunocompromised mice, suggest-
donor. Indeed, this vector and treatment portfolio has been ing their benign nature. Thus, the expansion of gp91 phox+ cells,
licensed in 2011 to a major pharmaceutical company for clinical although clearly providing therapeutic benefit during the initial
development. phase, was viewed with mixed feelings by the investigators and the
general gene therapy community.
More recent follow-up on these two study patients has been
Chronic Granulomatous Disease provided. 13,14 Indeed, although gene marking remained high in both
patients, downregulation of gene expression was noted as a result of
Chronic granulomatous disease (CGD) is an inherited disorder of CpG methylation in the viral LTR promoter. As a consequence,
phagocyte dysfunction characterized by often life-threatening invasive gp91phox expression was suppressed, but the capacity of the LTR-
fungal and bacterial infections and by granuloma formation in vital encoded enhancer to transactivate nearby genes remained intact. One
organs. CGD results from a mutation in one of four subunits of the patient died 2.5 years after therapy of severe sepsis. The second
reduced form of nicotinamide adenine dinucleotide phosphate patient developed monosomy 7 and myelodysplastic syndrome
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oxidase of phagocytes. The inability to form microbiocidal oxygen (MDS) and died after an unsuccessful unrelated donor BMT. Of
species renders the phagocytes unable to fight invasive infections. note, the EVI1 locus has previously been identified as a common
Almost 70% of CGD cases result from defects in the X-linked gene target of retroviral oncogenesis. EVI1, which is not detected in
encoding gp91 phox (X-CGD). With conventional therapy, including normal hematopoietic cells, has been associated with myeloid leuke-
lifelong antimicrobial prophylaxis and interferon-γ therapy, the yearly mia and MDS. The constitutive overexpression of EvI1 in mouse BM
mortality rate of X-CGD remains at 5%. HSCT is curative for cells has been shown to induce MDS in mice and data from the CGD
patients with a perfectly matched sibling donor but remains risky in trial suggest that dysregulated expression is associated with genomic
patients with active infections. Unrelated donor transplantations are instability, presumably contributing to the acquisition of additional
not routinely recommended. Thus, the development of a gene somatic mutations. Despite these molecular events, the infusion of
+
therapy approach that uses autologous HSC provides an important gene-corrected CD34 cells was highly effective with regard to clear-
therapeutic advance for this patient group. In previous clinical gene ing refractory pyogenic infections, raising the possibility of using gene
therapy trials conducted without myeloreductive conditioning, the therapy to bridge patients with refractory pyogenic infections into
engraftment level of gene-modified cells remained low. eligibility for allogeneic HSCT. A multiinstitutional international
In 2002, the German group of Grez and colleagues in Frankfurt, trial has recently opened in which the GP91 phox gene is expressed in
12a
Germany, initiated a gene therapy trial of X-CGD. The initial a lentivirus backbone via a chimeric myeloid internal promoter
patients received a mild immunosuppressive preparative regimen and (NCT01855685).
failed to engraft significant numbers of gene-modified cells. However,
2 years later, low-dose busulfan—modeled on the successful gene
therapy trial for ADA-SCID—was incorporated into the preparative Wiskott-Aldrich Syndrome
regimen, and additional patients were treated. This group of patients
has been followed with unprecedented sophistication by the prospec- WAS is an X-linked immunodeficiency caused by inactivating muta-
tive monitoring of integration sites that marks each hematopoietic tions in the WAS protein (WASP). WASP plays a regulatory role in
cell before transplantation and then allows the tracking of these cells cell signaling and cytoskeletal reorganization in hematopoietic cells.
in vivo. The initial two patients treated were 26 and 25 years old. The disease is fatal and is characterized by severe combined immu-
Both patients carried the diagnosis of X-CGD and had failed to clear nodeficiency, thrombocytopenia, elevated frequency of tumor forma-
invasive infections, including a Staphylococcus aureus liver abscess and tion, eczema, and other autoimmune manifestations. The only
pulmonary aspergillosis with medical treatment. Thus, autologous currently available curative therapy for WAS is BM transplant, but
+
peripheral blood CD34 cells were mobilized with granulocyte as with the other primary immunodeficiencies, the availability of
colony-stimulating factor and collected. Gene transfer was performed suitably matched donors is limiting. A clinical trial for the genetic
using a γ-retroviral vector SF71gp91 phox . This vector, containing the correction of WAS via retroviral delivery of the WASP cDNA into
+
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spleen focus-forming virus LTR elements (in contrast to the Moloney autologous CD34 cells was recently reported. A combination of a
+
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MLV LTR in the two SCID trials) was chosen for its ability to achieve relatively high cell dose (7–8 × 10 CD34 /kg body weight) and good
high expression levels in transduced HSCs. The in vitro transduction transduction efficiency led to gene marking across both myeloid and
rates in the two patients were 45% and 39.5%, respectively, with a lymphoid lineages. A marked clinical benefit from gene therapy has
15
proviral copy number of 2.6 and 1.5 per transduced cell. Proviral been reported in one of the patients. Of particular note is the fact
integration occurred preferentially in gene-coding regions (47–52%) that the architecture of the vector backbone used in this trial is similar
and was highly skewed toward the 5-kb sequence surrounding TSS. to that used in both the CGD and the SCID trials described earlier.
Moreover, the clonal distribution pattern was not stable over time. That is, the γ-retroviral vector has an intact LTR that contains the
16
Rather, starting 5 months after therapy, a less diverse integration enhancer and promoter from the spleen focus-forming virus. Recent
pattern emerged, indicating the appearance of dominant clones. reports describe T-cell leukemia from insertional mutagenesis in 7 of
17
Clinically, following a period of cytopenia after conditioning and the 10 patients in this trial. Thus, a common molecular etiology in
cell infusion, the initial engraftment rates detected in the peripheral this trial strongly suggests that the initial vector design (intact strong
blood were 12% to 13%. Significant improvement in the previously viral LTRs containing enhancer elements that can transactivate pro-
refractory infections was noted 50 to 60 days after therapy. Surpris- moters over long distances) are unsafe. In addition, the occurrence of
ingly, a gradual increase in the number of gene-corrected cells up to T-cell leukemia in a disease other than SCID weakens the argument
50% to 60% of all peripheral blood cells was observed, starting that the leukemias in X-SCID was related either to the transgene or
around day 150 after transplant. This coincided with increased some unknown disease-specific characteristic. In any regard, use of
oxidase activity and occurred in the absence of altered blood counts. the “first-generation” MLV backbone in human trials may now be
These events were accompanied by a selective outgrowth of progeni- ended. More recently, several new clinical trials have opened that
tors carrying vector insertions that activated one of three oncogenes: utilize a SIN lentivirus vector for treatment of WAS (NCT02333760,
PRDM16, SETBP1, and most notably MDS-EVI1. Although all NCT01410825, and NCT01347346). These trials utilize the same
three genes are well-known cancer-associated genes, most clonal vector backbone. In a total of 10 patients studied, 9 were reported
outgrowths were exhausted after a few months with the exception of alive and showed clinical benefit with reduced autoimmunity, includ-
MDS-EVI1, which increased to 67% to 90% in both patients ing improvement in eczema and reduced platelet transfusion depen-
approximately 1 year after cell infusion. Of note, the dominant dence with no serious bleeding. One patient died from preexisting
MDS-EVI1 clones initially did not transgress the boundaries of the viral infection that did not resolve after gene therapy. In one study it
normal myeloid pool because these cells remained cytokine dependent was noted that the degree of myeloid engraftment and platelet