Clinical Impact of Suicide Gene Therapy in Allogeneic Hematopoietic Stem Cell Transplantation

Abstract

Allogeneic hematopoietic stem cell transplantation (allo-SCT) from an HLA-matched related or unrelated donor is a curative option for patients with high-risk hematological diseases. In the absence of a matched donor, patients have been offered investigational transplantation strategies such as umbilical cord blood SCT or family haploidentical SCT. Besides the activity of the conditioning regimen, most of the antileukemic potential of allo-SCT relies on alloreactivity, promoted by donor lymphocytes reacting against patient-specific antigens, such as minor and major histocompatibility antigens, ultimately translating into cancer immunotherapy. Unfortunately, alloreactivity is also responsible for the most serious and frequent complication of allo-SCT: graft-versus-host-disease (GvHD). The risk of GvHD increases with the level of HLA disparity between host and donor, and leads to impaired quality of life and reduced survival expectancy, particularly among patients receiving transplants from HLA-mismatched donors. Gene transfer technologies are promising tools to manipulate donor T cell immunity to enforce the graft-versus-tumor effect, to promote functional immune reconstitution (graft vs. infection), and to prevent or control GvHD. To this purpose, several cell and gene transfer approaches have been investigated at the preclinical level, and are being implemented in clinical trials. Suicide gene therapy is to date the most extensive clinical application of T cell-based gene therapy. In several phase I–II clinical studies conducted worldwide this approach proved highly feasible, safe, and effective in promoting a dynamic and patient-specific modulation of alloreactivity. This review focuses on this approach.

Introduction

A llogeneic stem cell transplantation (allo-SCT) is the most effective curative option for high-risk hematological malignancies and selected inherited disorders (Appelbaum, 2008; Ljungman et al., 2009), and has been proposed as an investigational therapeutic approach to treat autoimmune diseases and solid tumors (Demirer et al., 2008; Gratwohl, 2008). The clinical efficacy of allo-SCT against malignancies relies on high-intensity chemotherapy and radiotherapy, and on the activity of the allogeneic immune system. In allo-SCT, donor immunity offers opportunities and challenges. The graft-versus-tumor (GvT) effect, which originates from the allogeneic immune system of the donor reacting against the underlying malignancy, represents the major immunological opportunity for allo-SCT and the most convincing clinical evidence of the potency of cancer immunotherapy. On the other hand, recognition of several healthy host tissues by alloreactive donor lymphocytes leads to a detrimental immune response: graft-versus-host disease (GvHD). Several clinical observations have clearly established the role of alloreactive lymphocytes in controlling cancer cells: (1) Depletion of donor lymphocytes from the graft, the most effective approach to prevent GvHD, is affected by a high incidence of disease relapse (Horowitz et al., 1990); (2) the occurrence of GvHD is highly associated with long-term complete remission of neoplastic diseases (Kolb, 2008), suggesting that GvHD and GvT are two different clinical manifestations of the same biological event, that is, the alloreactive response; and (3) immune editing of acute myeloid leukemia, relapsing after allo-SCT, indicates that alloreactive cells impose critical selective pressure on cancer cells. We showed that, in the context of haploidentical stem cell transplantation, leukemic blasts undergo genomic rearrangements that result in loss of the patient-specific HLA haplotype, the major target of alloreactivity, and that such rearrangement accounts for 30% of leukemia relapse events in this transplantation setting (Vago et al., 2009). Despite the high efficacy of allo-SCT, three principal limitations must be overcome for its full exploitation: transplant toxicity, donor availability, and GvHD. The toxicity of the conditioning regimen has been largely addressed by the use of new drugs with a reduced toxicity profile, thus permitting the extension of this procedure also to aged patients and to patients with comorbidities (Gratwohl et al., 2009). The lack of compatible donors is currently addressed by the implementation of transplantation strategies based on alternative donor grafts, such as cord blood or stem cells from haploidentical family members (haplo-SCT) (Aversa et al., 2008). To date, GvHD remains a major and unsolved complication of allo-SCT that significantly impairs the quality of life and the survival expectancy of allo-SCT patients (Deeg, 2007; Baker and Fraser, 2008). This complication is particularly problematic in the context of haplo-SCT, in which HLA antigen mismatch can lead to severe and life-threatening GvHD (Huang, 2008), and the extensive T cell depletion required to prevent this reaction leads to profound and prolonged posttransplantation immunodeficiency and high mortality (Ciceri et al., 2008). The tight association of the beneficial GvT effect with GvHD represents a major challenge, and encourages the design of novel strategies able to preserve and enhance posttransplantation immune reconstitution and GvT, while allowing the prevention or control of GvHD. In spite of the tremendous effort to develop an effective pharmacological prophylaxis and treatment of GvHD, its outcome still remains highly unsatisfactory. The first-line treatment of GvHD, consisting of high-dose steroids, results in less than 50% clinical responses. Despite the availability of several second-line treatments, including immunosuppressive drugs and monoclonal antibodies, the primary response to first-line treatment is the most relevant predictor of long-term survival among GvHD patients (Paczesny et al., 2009). In this complex setting, several approaches of cell therapy and gene therapy aimed at controlling GvHD while preserving GvT and immune reconstitution have been designed and tested.

Cell Therapy for GvHD

Stemming from the clinical observation that conventional pharmacological prophylaxis and treatment of GvHD are largely unsatisfactory, several approaches of cell therapy have been developed and tested in clinical trials. The common purpose of these strategies is to modulate the effects of donor immunity so as to restrain GvHD while boosting GvT and posttransplantation immune reconstitution. The majority of these approaches aim at reducing the alloreactive potential of donor lymphocytes, to spare nonalloreactive recall responses to pathogens, and putative nonalloreactive GvT effectors.

Infusion of mesenchymal stem cells

Because of their antiproliferative, immunomodulatory, and antiinflammatory effects, mesenchymal stem cells (MSCs) have been hypothesized as promising tools for the management of GvHD, and their immune regulatory effects are currently under intense investigation (Ramasamy et al., 2008; Tian et al., 2008). A phase I–II clinical study showed the safety and efficacy of the infusion of MSCs, in conjunction with immunosuppressive drugs, for the management of steroid-resistant GvHD (Le Blanc et al., 2008); however, results were not reproduced in subsequent studies (von Bonin et al., 2009), and two large phase III sponsored randomized studies failed to show advantages in the survival of patients affected by GvHD and treated with MSCs (Baker, 2009).

Depletion of selected T cell subsets

Several studies in murine allo-SCT models showed a differential contribution of specific T cell subsets in the pathogenesis of GvHD (Shlomchik, 2007). Initial studies pointed to CD8⁺ lymphocytes as major players in the pathogenesis of GvHD. In clinical trials, infusion of CD8⁺ cell-depleted grafts or CD8⁺ cell-depleted donor lymphocyte infusions (DLIs) produced mixed results in the context of HLA-identical SCT (Nimer et al., 1994; Giralt et al., 1995; Ho et al., 2004), possibly because of variable levels of CD8⁺ cell contamination. The infusion of DLI, highly depleted of CD8⁺ lymphocytes, induced a low (26%) rate of GvHD in haplo-SCT (Dodero et al., 2009). Murine models showed that naive T cells from unprimed donors induce lethal GvHD, whereas memory T cells do not induce GvHD but sustain a significant GvT effect (Chen et al., 2007; Zheng et al., 2008). In addition, several publications report on the role of regulatory T cells in modulating alloreactivity (Taylor et al., 2002; Edinger et al., 2003). Accordingly, refined graft manipulation procedures resulting in the administration of donor lymphocytes depleted of naive cells (Riddell and Appelbaum, 2007), or enriched for regulatory T cells (Sun et al., 2007; Nguyen et al., 2008), represent promising options for the prophylaxis of GvHD. Clinical trials exploring the safety and efficacy of these approaches are currently ongoing.

Selective depletion of alloreactive lymphocytes from the graft

Selective depletion from the graft of alloreactive lymphocytes aims at separating GvHD and GvT/GvI (graft vs. infection) reactivities through the administration of a wide but safe T cell repertoire selectively depleted of alloreactive cells. This is pursued through the in vitro stimulation of donor lymphocytes with host antigen-presenting cells, followed by the removal of activated—and thus alloreactive—lymphocytes before infusion. After stimulation with host cells, activated alloreactive lymphocytes may be identified by their surface phenotype (Amrolia et al., 2006; Wehler et al., 2007) or by preferential accumulation of photoactive dyes (Mielke et al., 2008; Perruccio et al., 2008), and sorted accordingly. Clinical trials exploring the efficiency of this approach in preventing GvHD are currently ongoing.

Modulation of innate immunity and inflammation

Dendritic cells (DCs) play a nonredundant role in the development of GvHD in murine models (Shlomchik et al., 1999). It was shown that the infusion of granulocyte colony-stimulating factor (G-CSF) increases the incidence of GvHD through the activation of host DCs, which upregulate G-CSF receptor expression after total body irradiation (Morris et al., 2009). Accordingly, treatment with antiinflammatory agents, such as histone deacetylase inhibitors, reduces the incidence and severity of GvHD in murine models and has been proposed to treat GvHD (Reddy et al., 2008).

Suicide Gene Therapy Approach to Control GvHD

The cell therapy approaches described previously aim at reducing the repertoire of donor lymphocytes infused with or after grafting, so to prevent alloreactivity. Thus, in these approaches, the antileukemic potential of allo-SCT is intrinsically depleted of one of its most potent mechanisms: alloreactivity. In contrast, the suicide gene therapy approach aims at providing transplanted patients with a wide and polyclonal population of donor T cells, inclusive of alloreactive specificities, to promote immune reconstitution and possibly exploit the alloreactive and nonalloreactive components of GvT. This approach is not designed to prevent GvHD, but to selectively control it, in patients in need, and the possibility to control the detrimental effect of alloreactivity allows full exploitation and, theoretically, exacerbation of the alloreactivity itself, with the ultimate goal of promoting GvT. The expression of a suicide gene confers to cells sensitivity to a molecule (prodrug) that is nontoxic to nonexpressing cells. In the case of donor lymphocytes the activation of the suicide system represents a tool for the selective control of alloreactivity (Tiberghien et al., 1994; Verzeletti et al., 1998). Once genetically modified to express a suicide gene, donor lymphocytes can be infused into cancer patients to cure/prevent disease relapse, or to control opportunistic infections. In the case of GvHD, the “suicide” machinery can be triggered in transduced cells by the administration of the prodrug, to selectively eliminate the infused genetically modified lymphocytes, leading to GvHD abrogation, without interfering with the natural process of posttransplantation immune reconstitution (Bonini et al., 1997; Tiberghien et al., 2001). Suicide gene therapy has been applied in several clinical trials in the context of allo-SCT, and the infusion of donor lymphocytes expressing a suicide gene represents the most extensive clinical application of a T cell-based gene therapy approach.

Theoretically, vectors, transgenes, and protocols used for T cell manipulation should be designed to avoid perturbations of T cell function, repertoire, and expansion abilities, and the resulting genetically modified cells should be similar to unmanipulated lymphocytes, except for the expression of the suicide molecule.

In the following sections critical issues for effective suicide gene therapy, such as vectors, T cell manipulation protocols, and types of suicide genes, are discussed.

Vectors, T Cell Transduction Protocols, and Selection Procedures

The successful clinical application of suicide gene therapy in allo-SCT relies on efficient control of GvHD, and thus on the ability to homogeneously infuse large numbers of lymphocytes stably expressing the suicide gene at high levels. This greatly depends on the efficiency of the vector and on the use of strong promoters, most frequently the retroviral long terminal repeat (LTR), enabling stable, high-level suicide gene expression in target cells. In addition, clinical results of pilot studies with genetically modified lymphocytes underlined the importance of a minimal ex vivo manipulation procedure, not only to reduce costs and risks of contamination but also to preserve the in vivo expansion potential of gene-modified cells (Ferrand et al., 2000; Marktel et al., 2003). This is crucial to promote long-term persistence of gene-modified T cells and to sustain the antileukemic effect.

The current clinical application of suicide gene therapy in allo-SCT relies on retroviral vectors (RVs). RV-mediated T cell transduction requires T cell proliferation, usually obtained with anti-CD3 monoclonal antibodies: T cell receptor triggering induces T cell activation and proliferation, thus permitting efficient transduction, but results in two major limitations: (1) loss of T cell precursors (Sauce et al., 2002) and (2) differentiation of transduced lymphocytes toward an effector/effector memory functional phenotype, characterized by low expansion capacity, a short expected half-life, and low alloreactive potential (Bondanza et al., 2006). By reducing alloreactivity this procedure may limit the antileukemia effect mediated by transferred lymphocytes. In contrast to effector cells, central memory lymphocytes (T_CM) display a high proliferative and expansion potential and proved superior to effector and effector memory cells in promoting cancer regression in murine models (Klebanoff et al., 2005).

Our group proposed a gene transfer protocol based on CD28 costimulation and T cell culture with a combination of interleukin (IL)-7 and IL-15 to generate suicide gene-modified T cells with a central memory functional phenotype. These cells are characterized by a high expansion potential and alloreactivity profile, comparable to that of unmanipulated lymphocytes. Alloreactivity mediated by suicide gene-expressing T_CM cells can be controlled through activation of the suicide machinery (Kaneko et al., 2009), thus allowing safe exploitation of the potency of alloreactive cells against cancer.

To overcome the need for T cell activation, and the subsequent drive toward terminal T cell differentiation, lentiviral vectors (LVs) can be employed to introduce the suicide gene into T cells. LVs efficiently transduce nondividing cells by active transport of the viral preintegration complex into the nucleus (Bukrinsky et al., 1992). Although resting T cells are not permissive to HIV or to lentiviral transduction, culture with low doses of γ-chain cytokines allows efficient LV-mediated gene transfer into human lymphocytes, in the absence of T cell receptor (TCR) triggering, resulting in the preservation of an early (naive and central memory) T cell phenotype (Cavalieri et al., 2003; Verhoeyen et al., 2003; Qasim et al., 2007). Novel lentiviral vectors pseudotyped with measles glycoproteins have been generated to further reduce the extent of T cell manipulation, thus permitting efficient gene transfer in resting lymphocytes in the absence of exogenous cytokines (Frecha et al., 2008). Lentiviral vectors have entered the clinical investigation phase, have been proved safe, and have produced promising clinical results (Levine et al., 2006; Cartier et al., 2009), opening the field to novel applications. Because retroviral vectors were shown to be safe and effective when applied to T lymphocytes, the high transduction efficiency obtained with lentiviral vectors, under minimal culture conditions, has encouraged their clinical implementation.

Selection of gene-modified cells, aimed at isolating a relatively pure population of transduced lymphocytes before infusion into patients, is an essential step for efficient control of GvHD. The most widely used selection marker is represented by a truncated form of the low-affinity receptor for human nerve growth factor (Mavilio et al., 1994). The cell surface selectable marker gene is currently included in vectors to ensure selection and in vivo tracing of transduced cells. Alternative cell surface markers have been proposed and tested in preclinical models: among these, CD34 has the advantage of allowing selection of transduced cells by exploiting a well-validated GMP-grade selection platform, extensively used in the allo-SCT setting (Fehse et al., 2000). A fusion HSV-TK/CD34 gene has been developed to ensure the suicide phenotype in selected cells (Fehse et al., 2002). However, posttranslational breakage of the fusion protein, leading to a loss of the suicide phenotype in transferred cells, was shown (Bennour et al., 2008).

Suicide Gene/Prodrug Systems

To date the suicide gene prototype, and the only system validated in clinical trials, is the herpes simplex virus thymidine kinase (HSV-TK) gene that confers ganciclovir sensitivity to transduced cells. Several phase I–II clinical trials involving more than 120 patients showed the safety of the TK-based gene therapy approach (Bonini et al., 2007). Transduced HSV-TK-expressing cells (TK cells) phosphorylate the prodrug ganciclovir, leading to accumulation of cytotoxic antimetabolites that induce cell death. When applied to HLA-identical and haploidentical hematopoietic stem cell transplantation the HSV-TK/ganciclovir system proved safe and efficient in promoting selective control of GvHD. Despite these encouraging results, the broad clinical application of the TK approach led also to the identification of three major limitations: (1) the presence of an alternative splicing site in the TK gene, resulting in a nonfunctional TK enzyme (Garin et al., 2001); (2) the unwanted elimination of modified T cells in the case of cytomegalovirus (CMV) reactivation, requiring treatment with ganciclovir; and (3) the immunogenicity of viral epitopes encoded by the viral HSV-TK transgene, resulting in the unwanted elimination of gene-modified cells (Riddell et al., 1996). To address the first limitation, mutated TK gene variants devoid of cryptic splicing sites were developed and validated at the preclinical level to avoid alternative splicing (Chalmers et al., 2001; Kaneko et al., 2009). On the second issue, clinical experience clearly showed that the need for ganciclovir to treat CMV is highly counterbalanced by the wide immune reconstitution promoted by TK cells, ultimately leading to CMV control (Ciceri et al., 2009). Immunogenicity of TK has been claimed as a major obstacle. Immunity against TK occurs mainly when gene-modified cells are infused into immune-reconstituted patients (Berger et al., 2006) and, overall, does not prevent the exploitation of the antitumor potential of this strategy when applied to immunodeficient patients (Traversari et al., 2007; Mercier-Letondal et al., 2008). Several novel suicide genes encoding for human, and possibly nonimmunogenic, proteins have been proposed and tested at the preclinical level to overcome the immunogenicity of HSV-TK (Table 1).

Table 1.

Suicide Genes in Perspective

Suicide gene/prodrug	Advantages	Disadvantages
HSV-TK/GCV	• Safe in several allo-SCT clinical settings • Effective abrogation of GvHD by the suicide/prodrug system in all clinical trials • Extensive clinical experience with use of the prodrug	• Immunogenicity of the HSV-TK protein in immunocompetent patients • Unwanted elimination of modified T cells in case of CMV treatment with GCV (but not with foscarnet)
CD20/anti-CD20 mAb	• Human protein not immunogenic in humans • No additional selection marker required • Extensive clinical experience with use of the prodrug	• Not yet tested in clinical trials • Unwanted depletion of B cells by anti-CD20 mAb • Unwanted elimination of gene-modified T cells by the treatment of CD20⁺ tumors with anti-CD20 mAb • High expression levels of CD20 required for cell death
FKBP-FAS/AP20187, AP1903 dimerizers	• Human components with low immunogenic potential in humans • Prodrug with a high specificity profile	• Limited clinical experience with the prodrug • Suicide system efficiency limited in cells expressing antiapoptotic molecules • Suicide system not tested in clinical trials
FKBP-caspase-9/AP20187 dimerizer prodrug	• Human components with low immunogenic potential in humans • Prodrug with a high specificity profile • Suicide system unaffected by the overexpression of antiapoptotic molecules	• Limited clinical experience with the dimerizer • Clinical efficacy of the suicide system not yet reported

Abbreviations: allo-SCT; allogeneic hematopoietic stem cell transplantation; CMV, cytomegalovirus; FKBP, FK506-binding protein; GCV, ganciclovir; GvHD, graft-versus-host disease; HSV-TK, herpes simplex virus thymidine kinase.

One of the first human suicide genes developed was based on CD20, a cell surface molecule that renders T cells sensitive to anti-CD20 antibodies, drugs largely and successfully used to treat CD20⁺ B cell malignancies. The major advantages of CD20 include the lack of immunogenicity in humans and the ability to use the same suicide molecule as a marker for immune selection of transduced cells (Introna et al., 2000). In vitro studies showed that CD20 expression does not interfere with T cell function (Serafini et al., 2004), but variable levels of transgene expression were shown to affect the efficacy of the suicide gene/prodrug system. Heemskerk and colleagues proposed the use of CD20 as a safety switch to control the possible autotoxicity that may result from TCR mispairing on the transfer of a tumor-specific TCR in human lymphocytes, and showed efficient killing of CD20-expressing cells (Griffioen et al., 2009).

Among novel, nonimmunogenic suicide genes, a chimeric gene incorporating the death domains of caspase-9 (iCasp9) was developed by Brenner and colleagues. The molecule is fused to a modified human FK506-binding protein, so that cell killing is elicited by a nontoxic chemical inducer of dimerization. When transferred into human lymphocytes, depleted of alloreactive specificities, iCasp9 allowed efficient T cell killing by the dimerizer. Engineered cells retained the ability to mediate memory immune responses against pathogens, and contained a subset of T cells with regulatory phenotype and function. Transgene expression was downregulated in quiescent T cells, but promptly upregulated on activation (Tey et al., 2007). The efficacy of the iCasp9/dimerizer system was successfully tested in a murine model of cell therapy-induced type I diabetes (de Witte et al., 2008) and a clinical trial investigating the safety and efficacy of iCasp9-transduced donor lymphocytes to control GvHD is currently ongoing.

Clinical results of suicide gene therapy in hematopoietic stem cell transplantation

Suicide gene therapy applied to allogeneic hematopoietic stem cell transplantation has been tested in several phase I–II clinical trials in Europe, the United States, and Japan and is one of the first cell-based gene therapy approaches to be tested in the context of a randomized phase III clinical trial, designed to evaluate the efficacy of TK cells in reducing mortality subsequent to transplantation. The safety and activity of the TK-based gene therapy approach was shown in 123 patients (Table 2). Approximately 50% of treated patients obtained clinical benefit, although clinical responses varied in different trials, possibly because of differences in transplantation settings, vectors, and manipulation procedures. The average rate of GvHD was 21% and, most importantly, every case of GvHD occurring after the infusion of TK cells was completely controlled, indicating the efficacy of the suicide gene/prodrug system in taming alloreactivity.

Table 2.

Clinical Trials of Suicide Gene Therapy in Allogeneic Hematopoietic Stem Cell Transplantation

Clinical application	Vector (suicide gene/marker gene)	T cell manipulation (days)	No. of TK cells infused/kg per patient	No. of treated patients	Clinical response (no. of patients)	Incidence of GvHD (no. of patients)	Complete response of GvHD to GCV	Reference
To treat disease relapse occurring after HLA-identical allo-SCT	RV (HSV-TK/ΔLNGFr)	14	10⁵–10⁸	23	11	4	3/3^a	(Bonini et al., 1997); (Ciceri et al., 2007)
	RV (HSV-TK/NeoR)	NE	10⁶–10⁸	23	6	0	NE	(Champlin et al., 1999)
	RV (HSV-TK/NeoR)	NE	10⁶	3	1	1	NE	(Munshi et al., 1997)
	RV (HSV-TK/NeoR)	24–48	10⁶–10⁸	9	2	1	1/1	(Burt et al., 2003)
	RV (HSV-TK/ΔLNGFr)	9–11	10⁸	5	4	2	2/2	(Onodera, 2008)
Day 0 in TCD allo-SCT	RV (HSV-TK/NeoR)	12	10⁵–10⁶	12	4	5	5/5^b	(Tiberghien et al., 2001)
	RV (HSV-TK/NeoR)	13	5 × 10⁶	3	1	2	1/1	(Fehse et al., 2004)
Day 60 in TCD allo-SCT	RV (HSV-TK/ΔLNGFr)	10	10⁷	9	7	1	NE	(Weissinger et al., 2008)
Day 42 in TCD haplo-SCT	RV (HSV-TK/ΔLNGFr)	14	10⁵–10⁷	8	3	1	1/1	(Bonini et al., 2007)
	RV (HSV-TK/ΔLNGFr)	10	10⁷	28	22	11	10/10^c	(Ciceri et al., 2009)
Total				123	61	28	23/23

Abbreviations: GCV ganciclovir; GvHD, graft-versus-host disease; LNGF, low-affinity nerve growth factor; NE, not evaluable; RV, retroviral vector; SCT, stem cell transplantation; TCD, T cell depleted; TK, thymidine kinase.

One patient with GvHD achieved complete response (CR) after GCV administration and immunosuppressive drugs.

One patient with GvHD achieved CR after administration of GCV and steroids.

Four patients with GvHD achieved CR after administration of GCV and short course low-dose steroids, and two patients with GvHD achieved CR after GCV administration and immunosuppressive drugs.

In the context of HLA-identical allo-SCT, TK cell infusion proved feasible and promoted GvT activity directly correlated with the level of in vivo expansion of gene-modified lymphocytes. In this setting, we treated, by multiple infusions of TK cells, 23 patients experiencing recurrence of hematological malignancies occurring after allo-SCT. Seventeen patients were evaluable for engraftment and GvT, and 11 of 17 patients experienced clinical benefit resulting in 6 complete remissions and 5 partial responses. Four patients developed GvHD, and three of four required treatment with ganciclovir with successful control of all symptoms.

Tiberghien and colleagues developed a protocol of suicide gene therapy associated with T cell-depleted bone marrow transplant from HLA identical sibling donors. Twelve patients received TK cells on day 0 posttransplantation according to an escalating dose protocol. Four patients experienced GvHD, obtaining complete remission in all cases through exploitation of the suicide machinery (Tiberghien et al., 2001). Starting from the results obtained in the HLA identical setting, we translated the suicide gene approach in the context of SCT from haploidentical family donors, to induce early immune reconstitution and promote GvT while selectively controlling GvHD. In a first pilot experience we treated eight patients with HSV-TK lymphocytes after CD34-selected haplo-SCT, to promote immune reconstitution and GvT while preserving tight control of GvHD. Three of eight patients were evaluable for HSV-TK-induced immune reconstitution, and one patient developed GvHD and was successfully treated with ganciclovir (Bonini et al., 2007). More recently, we confirmed the safety and efficacy of the suicide gene transfer technology applied to haplo-SCT in abrogating late transplantation-related mortality, a highly frequent event in the context of T cell-depleted haplo-SCT (Ciceri et al., 2008). In a phase I–II multicenter trial, sponsored by MolMed (Milan, Italy), TK cells were infused into patients undergoing haplo-SCT. Fifty patients were transplanted with T cell-depleted grafts from haploidentical donors for high-risk leukemia and 28 patients received TK cells 1 month after SCT. Twenty-two patients obtained prompt immune reconstitution with more than 100 circulating T lymphocytes per microliter within the first 3 months after haplo-SCT. Late infectious mortality was completely abrogated, suggesting that the infusion of gene-modified lymphocytes promoted rapid and protective immune reconstitution. Ten patients developed acute GvHD (grades I to IV) and 1 developed chronic GvHD, always controlled by suicide gene induction (Ciceri et al., 2009).

Long-term persistence of TK cells has been reported by various authors, suggesting that infusion of gene-modified lymphocytes might mediate long-term immunosurveillance (Recchia et al., 2006; Deschamps et al., 2008). The safety of the TK-based gene therapy approach has been documented by a large number of preclinical studies, and by phase I–II clinical trials: no adverse or toxic event related to the gene transfer procedure has been reported to date (Bonini et al., 2003). Analyses of integrated proviruses identified in lymphocytes confirmed preferential integration of retroviral vectors near transcription start sites of active genes (Recchia et al., 2006; Deschamps et al., 2008). Comparative retrospective analyses of transduced cells harvested before and after infusions into patients suggest that integrations interfering with T cell function lead mainly to clonal ablation. Remarkably, suicide gene-expressing lymphocytes maintain stable gene expression profiles, immune competence, and a wide repertoire in vivo for more than 10 years after administration, with no evidence of clonal selection.

Conclusions and Remarks

Suicide gene therapy applied to allogeneic hematopoietic stem cell transplantation has emerged as a promising option to spare the beneficial GvT and GvI effects of donor lymphocytes from detrimental GvHD. In several clinical phase I and II studies conducted worldwide this approach proved highly feasible, safe, and active in promoting dynamic and patient-specific modulation of alloreactivity. A registrative, multicenter, randomized phase III study has already begun in Europe to verify the impact of gene therapy in improving overall survival of cancer patients. If successful, this trial will lead to the registration of a gene therapy approach as a cell-based medicinal product, a critical step for gene therapy to finally establish its role as a major therapeutic tool. From this perspective, a major effort will be required to contain costs and prove proper sustainability.

Acknowledgments

This work was supported by grants from the Italian Ministry of University and Research, from the Italian Ministry of Health, and from the Italian Association for Cancer Research (AIRC).

Author Disclosure Statement

No competing financial interests exist.

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