Successes and risks of gene therapy in primary immunodeficiencies

Series editors: William T. Shearer, MD, PhD, Lanny J. Rosenwasser, MD, and Bruce S. Bochner, MD

Successes and risks of gene therapy in primary immunodeficiencies Javier Chinen, MD, PhD, and Jennifer M. Puck, MD Bethesda, Md This activity is available for CME credit. See page 40A for important information.

Several primary immunodeficiencies are under consideration for gene therapy approaches because of limitations of current standard treatment. Many primary immunodeficiencies are caused by defects in single genes expressed in blood cells; thus addition of a correct copy of the gene to hematopoietic stem cells (HSCs) can generate immune cells with restored function. HSCs can be removed from a patient, treated outside the body, and reinfused. In the last decade, significant improvements have been made in transferring genes by means of retroviruses to HSCs in vitro, and gene therapy trials for patients with Xlinked severe combined immunodeficiency (XSCID) and adenosine deaminaseedeficient severe combined immunodeficiency have restored immune competence. Gene therapy is actively being pursued in other immunodeficiency disorders, including chronic granulomatous disease and Wiskott-Aldrich syndrome. However, enthusiasm for the correction of XSCID by means of gene therapy has been tempered by the occurrence of 2 cases of leukemia in gene therapy recipients caused by insertion of the retroviral vector in or near the oncogene LMO2. The likelihood of retroviral insertional mutagenesis was estimated to be very low in the past on the basis of theoretic calculations and the absence of observed malignancies in animal studies and early clinical trials. Emerging new findings on retroviral integration both in the patients with XSCID and experimental animals now indicate that the insertion of retroviral sequences into the genome carries significant risk. Understanding the magnitude of risk is now a priority so that safety can be improved for future gene therapy clinical trials. (J Allergy Clin Immunol 2004;113:595-603) Key words: Primary immunodeficiency, gene therapy, insertional mutagenesis, retroviral vector, LMO2, X-linked severe combined immunodeficiency, adenosine deaminase, severe combined immunodeficiency

Primary immunodeficiencies (PIs) comprise a heterogeneous group of heritable diseases characterized by a dysfunctional or nonexistent immune response. The From the Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health. Disclosure of potential conflict of interest: J. Chinen—none. J. M. Puck—none. Received for publication January 2, 2004; revised January 5, 2004; accepted for publication January 20, 2004. Reprint requests: Jennifer M. Puck, MD, Genetics and Molecular Biology Branch, National Human Genome Research Institute, NIH Bldg 49, Rm 4A14, 49 Convent Dr, Bethesda, MD 20892. 0091-6749 doi:10.1016/j.jaci.2004.01.765

Abbreviations used ADA: Adenosine deaminase BMT: Bone marrow transplantation BTK: Bruton tyrosine kinase CD40L: CD40 ligand CGD: Chronic granulomatous disease GVHD: Graft-versus-host disease HSC: Hematopoietic stem cell IVIG: Intravenous immunoglobulin LAD: Leukocyte adhesion deficiency PEG: Polyethylene glycol PI: Primary immunodeficiency SCID: Severe combined immunodeficiency WAS: Wiskott-Aldrich syndrome WASP: WAS protein XLA: X-linked agammaglobulinemia XSCID: X-linked severe combined immunodeficiency

molecular and genetic basis of over 100 distinct PIs has been discovered in the past decade, and the list of disease genes for immunologic defects continues to grow.1-3 For example, 12 different genotypes have been identified for severe combined immunodeficiency (SCID; Table I).4-8 However, advances in finding the causes and explaining the pathophysiology of PIs have not yet been paralleled with similar progress in the development of safe and curative treatments. Patients with immunodeficiency have experienced considerable benefit from medical advances in supportive therapy, including the prophylaxis and treatment of infections, the management of chronic respiratory and gastrointestinal diseases, and the treatment of secondary neoplasias.9 However, the only definitive treatment available for the most serious PIs has been bone marrow transplantation (BMT). In 1968, the first successful BMT for immunodeficiency was reported.10 Bone marrow from an HLA-identical sibling was used to reconstitute a patient with X-linked SCID (XSCID). Although most patients do not have an HLA-matched related donor, almost every infant with SCID can receive an HLA-haploidentical BMT from a parent, provided the donor marrow is depleted of mature T cells that could react against the infant’s tissues.11 If the recipient is completely deficient in cellular immunity, preBMT ablative chemotherapy to avoid rejection is not 595

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TABLE I. Mutations in several distinct genes are responsible for SCID Reference

Defects in cytokine receptors and cytokine signaling cc deficiency, X-linked SCID JAK3 deficiency IL-7 receptor a chain deficiency CD45 deficiency CD3 d chain deficiency Defects in recombination of the antigen receptor genes of B and T cells Recombinase activating gene 1 (RAG1) deficiency Recombinase activating gene 2 (RAG2) deficiency Artemis deficiency Defects in purine pathway enzymes ADA deficiency Purine nucleoside phosphorylase deficiency Defects in modifiers of gene expression underlying multisystem disorders Cartilage hair hypoplasia SCID with alopecia and nail dystrophy (nude mouse ortholog)

1,2 1,2 1,2 1 6

1,2 1,2 1 1,2 1,2

7 8

required.12 BMT treatment can rescue up to 75% to 90% of infants given a diagnosis of SCID12-14 and is even more effective for infants proved to have SCID and undergoing transplantation in the first 3 months of life before devastating infections have set in.15 However, a significant proportion of patients who receive BMT achieve only partial or temporary immune reconstitution. BMT protocols for SCID in some immunodeficiency centers include the use of myeloablative conditioning of the recipient before the transplantation to improve engraftment. Myeloablation is designed to reduce the number of host stem cells present in the bone marrow that could compete with donor cells and decrease engraftment; however, myeloablation has unpredictable and potentially severe acute and late toxicity.14,16 Current approaches to donor cell purification and host treatment with chemotherapy for pretransplant conditioning and GVHD prophylaxis differ between transplant centers. In addition, the optimal treatment might be different for infants with distinct gene defects underlying their SCID and different environmental exposures. Multicenter studies of diseaseassociated variables, BMT treatment protocols, and longterm outcome are needed. BMT from HLA-matched sibling donors has been used with success to treat other PIs in addition to SCID, including lymphocyte disorders, such as Wiskott-Aldrich syndrome (WAS), hyper-IgM syndrome type 1, X-linked lymphoproliferative syndrome, and chronic granulomatous disease (CGD).17 Patients with these conditions have enough cellular immunity to reject allogeneic grafts unless cytoreductive conditioning is used, and those without an HLA-identical sibling currently face high risks from haploidentical or unrelated donor BMTs. Nonetheless, the

fact that providing normal hematopoietic cells can reverse these disease processes is an important prerequisite for consideration of gene therapy. Hematopoietic stem cells (HSCs) residing in the bone marrow are capable of lifelong self-renewal and differentiation on demand into all the hematopoietic lineages. Therefore disease states in which the pathology lies in the HSCs or in the cell lineages derived from HSCs can be cured by providing a healthy HSC population. Significant problems with allogeneic BMT can include failure to permanently engraft HSCs to give rise to a lifelong supply of all lymphoid lineages and graftversus-host disease (GVHD). Although new donor T cells emerge in patients with SCID approximately 90 days after BMT, it might take 2 years or more to develop production of immunoglobulins by B cells. More than half of the patients treated with haploidentical T celledepleted BMT fail to achieve sufficient reconstitution of the B-cell compartment to be able to stop receiving antibiotic prophylaxis and intravenous immunoglobulin (IVIG).12-14 Normal natural killer cell function after BMT might also be elusive. Patel et al18 have shown decreases in T-cell production and diversity in many patients with SCID several years after haploidentical BMT, suggesting that true stem cells might not be established from the donor. Moreover, some postBMT patients with SCID continue to experience frequent infections, growth failure, autoimmune disorders, and chronic lung disease.19 GVHD, another major limitation of BMT, might occur despite T-cell depletion in cases in which the donor is a haploidentical parent or a matched unrelated individual.12-14 Gene therapy has been investigated as an alternative to BMT for correction of genetic defects in HSCs without the risk of GVHD or graft rejection. Gene therapy is attractive for PIs for several reasons. Most PIs are monogenic; that is, mutations in a single gene lead to manifestations of the disease. Many of the genes responsible for PIs have been identified, and their DNA sequences and physiologic roles are known. The protein products of PI genes are highly expressed or even exclusively expressed in the hematopoietic system. Therefore, as demonstrated by successful treatment of PIs with allogeneic BMT, addition of a correct copy of the gene to HSCs can be expected to generate a continuous supply of immune cells with restored function. A further important feature of PI diseases is the facility with which the target cells for gene correction, HSCs, can be removed from a patient, enriched by means of cell selection, treated outside the body, and reinfused intravenously to home to the bone marrow. HSCs can be recovered from bone marrow samples, cord blood, or peripheral blood by means of apheresis after mobilization with granulocyte colony-stimulating factor20 or chemokines.21 Protocols to transduce human HSCs with retroviral vectors have significantly improved in efficiency over the past decade.22,23 After many years of vector development, animal studies, and human clinical trials, the successful treatment of human disease with gene therapy was first reported in 2 patients with XSCID in 2000.24 However, by the end of

2002, 2 of 10 infants who had received this treatment had T-cell leukemia.25 These unexpected adverse events have prompted investigators and regulatory agencies to reassess the risks of clinical trials for the gene correction of HSCs. We review the advances of gene therapy for PIs, along with current knowledge regarding the risks of insertional mutagenesis and leukemia.

PROGRESS OF GENE THERAPY FOR PRIMARY IMMUNODEFICIENCIES Adenosine deaminaseedeficient SCID The first gene that was identified to be defective in some patients with SCID encodes the enzyme adenosine deaminase (ADA). This purine pathway enzyme is present in every cell in the body, but its deficiency is primarily evident in lymphocytes because the purine intermediates that build up in the absence of ADA are extremely toxic to both B and T cells.26 In 1986, it was shown that murine retroviral vectors constructed to carry the ADA cDNA could correct ADA-deficient murine HSCs and human T cells in vitro.27,28 Thus ADA deficiency became the first genetic disorder in which the proof of concept of gene therapy was demonstrated. In 1990, ADA-deficient SCID became the first single-gene disease in a human clinical trial. Two ADA-deficient patients with SCID who had low numbers of T cells had some of their T cells removed, purified, and expanded ex vivo in IL-2. While in culture the cells were infected with a replication-incompetent retrovirus carrying the ADA cDNA. Transduced cells were reinfused into the subjects, in whom new ADA expression could subsequently be detected for up to 12 years.29,30 The patients experienced no ill effects and have remained in stable condition. However, the gene transfer was not efficient enough to produce clinical benefit. The concurrent administration to the patients of ADA stabilized by conjugation to polyethylene glycol (PEG-ADA) as a replacement enzyme for patients with ADA-deficient SCID31 added a factor that might have interfered with detecting clinical improvement. PEG-ADA therapy significantly increases the numbers of T and B cells of most patients with ADA deficiency and therefore removes the selective advantage that the gene-corrected cells might have. Among the important observations arising from this trial were the documentation of the longevity of correction in transduced and differentiated T cells (one patient still carries the transduced gene in approximately 20% of her peripheral blood lymphocytes),30 as well as the development of antibodies to FCS that were used during the ex vivo culture.32 FCS has been omitted from subsequent trials. During the past decade, several new developments in vector construction and methods for retroviral transduction of human HSCs have made incremental improvements in the gene-transfer efficiency of ADA retroviruses in clinical trials (Table II).22 After the initial ADA T-cell gene therapy trial, several clinical trials to attempt gene correction of ADA-deficient HSCs were undertaken.33-36 These resulted in expression of ADA in

TABLE II. Advances in retroviral (oncoretrovirus) gene transfer to HSCs d

d d

d

d

d

d

Retrovirus packaging cell lines with very low probability of generating replication-competent virus. Serum-free media to culture human HSCs. Pseudotyping with viral envelopes other than the original amphotrophic envelope (Gibbon ape leukemia virus envelope and feline type C virus RD114 envelope) that have more abundant receptors on human HSCs. Improved technology for HSC enrichment by selecting for the CD34 surface marker. Activating cytokines (Flt3 ligand, thrombopoietin, stem cell factor, IL-3, IL-6). Fibronectin-coated culture surfaces to improve transduction efficiency. Clinical scale vector production and transduction systems.

peripheral blood lymphocytes and, to a lesser extent, in other lineages for varying amounts of time, but no dramatic clinical benefit was achieved. Because ADA enzyme replacement was most likely decreasing the survival advantage of gene-corrected cells, PEG-ADA was progressively removed in 3 ADA-deficient patients who had received autologous genecorrected umbilical cord CD34+ cells. ADA-expressing T cells increased significantly during the months after withdrawal of PEG-ADA. However, PEG-ADA could not be completely discontinued because one of the patients had oral thrush, suggesting immunodeficiency.36 Recently, a ground-breaking study of ADA-deficient SCID was conducted by Aiuti et al,37 who performed gene therapy preceded by nonmyeloablative conditioning in the absence of any PEG-ADA therapy. Four patients were treated with subablative doses of chemotherapy, followed by infusion of autologous bone marrow CD34+ cells that had been transduced with a retrovirus carrying ADA. After a follow-up period ranging from 10 to 35 months, all patients were reported to be healthy. They have had normal numbers of T cells with proliferative responses to mitogens and antigens. Serum immunoglobulin levels and antibody responses to antigens have also become normal. The speed and degree of engraftment and immunoreconstitution were correlated with the dose of transduced CD34+ cells; in addition, the 2 patients who experienced the most myelosuppression had higher proportions of gene-corrected cells after the treatment.38 Because 2 modifications to previous approaches were made in this trial, it is not clear whether either the cytoreductive conditioning alone or the withholding of PEG-ADA alone would be sufficient to achieve a successful immune reconstitution. Despite the risks of the cytoreductive chemotherapy, this trial is the first to show complete reversal of the immunodeficiency of patients with ADA-deficient SCID.

XSCID In 1993, the disease gene for XSCID, the most common genetic form of SCID, was found to be IL2RG, which

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TABLE III. X-linked SCID as a target disease for gene therapy Advantages d Immunologic complications such as graft vs host disease, as seen after allogeneic BMT, are unlikely because of the autologous nature of this treatment. d The disease gene product is widely expressed in blood lineages. d Overexpression of cc is apparently not harmful. d There is an in vivo selective advantage for corrected lymphoid cells. d Target cells for correction are hematopoietic stem cells, which can be removed, transduced with retrovirus, and then reinfused. d Immune reaction against gene-corrected cells is unlikely because of the SCID phenotype. d Gene therapy in XSCID animal models has been successful. d Human gene therapy trials have provided full immune reconstitution to infants with XSCID. Disadvantages d It is not possible to predict or influence where retroviral vectors will integrate into the host DNA. d Retroviral insertion might inappropriately activate or inactivate host genes. d Clonal leukemic proliferation of transduced cells has occurred in 2 of 10 infants with XSCID treated with gene therapy in the French trial (no further cases have been detected >1 year after the initial 2 cases). d These were the youngest infants (1 and 3 months old at treatment). d Clonal proliferations were detected about 2½ years after gene therapy.

encodes the IL-2 receptor c chain.39,40 Subsequent studies revealed that this protein, now called the common c (cc) chain, is also part of the receptors for IL-4, IL-7, IL-9, IL-15, and IL-21.41 Male subjects, who have a single X chromosome, are affected by XSCID if they have a mutation of IL2RG, whereas female subjects, with 2 X chromosomes, can transmit the defect to their offspring but have healthy immune systems. Patients with XSCID lack T and natural killer cells and have functionally impaired B cells.41 XSCID is a good candidate for gene therapy for several reasons (Table III). There is a natural selective advantage for cells expressing cc, demonstrated by skewed X chromosome inactivation in lymphocytes from female carriers42 and a spontaneous reversion of a mutation associated with clinical improvement.43 In preclinical studies retroviral transduction of IL2RG to Bcell lines from patients with XSCID restored normal cc expression and signaling in response to IL-2 and IL-4.44-46 Gene transfer of IL2RG to HSCs from mice with XSCID restored normal immune function.47,48 Stem cells from patients with XSCID transduced with IL2RG differentiated into T and B cells in a chimeric sheep model.49 Human gene therapy trials for XSCID have achieved impressive immune reconstitution.24,50,51 In the group of Fischer et al from the Necker Hospital, Paris, France, infants with mutation-proved XSCID and no HLAidentical related donors were eligible for IL2RG gene transfer to autologous bone marrow cells, using a replica-

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tion-incompetent, murine leukemia virusederived retroviral vector. Transduced cells were reinfused without myeloablative treatment or immunosuppression. Nine of the 10 infants had new T cells with the transduced IL2RG.25,52 The single patient who did not experience immune reconstitution was very ill with disseminated BCG infection and had an enlarged spleen that the investigators hypothesized might have trapped the genecorrected cells. This patient later received a haploidentical BMT and is alive and well. All of the successfully genecorrected infants did well clinically, with development of retrovirus-bearing, cc+ T cells that were polyclonal and had normal proliferative responses to mitogens and antigens. Corrected B and natural killer cells were also detected. Importantly, in view of the poor attainment of antibody responses in many patients with XSCID after allogeneic BMT, the gene therapy recipients had normal titers of antibody to childhood vaccines and have not required continuation of IVIG. Expression and function of cc have persisted thus far as long as 4 years after the therapy, suggesting that hematopoietic progenitors have been transduced to ensure a long-term correction,25,50,52 but long-term durability (HSC correction) of the gene therapy treatment will require decades to prove. Thrasher,51 in England, observed a comparable immune reconstitution in 4 infants with XSCID treated with a gene therapy protocol that was similar to the French protocol. Because the French XSCID trial preceded the successful ADA trial discussed above, XSCID became the first human disease to be successfully treated with gene therapy as the sole treatment. Unfortunately, however, the 2 youngest infants in the French (1 and 3 months of age at treatment) trial experienced leukemic expansions of T-cell clones 30 and 34 months after infusion of corrected cells (see below).25,52 Both patients received antileukemic chemotherapy, and one received an allogeneic BMT when his response to chemotherapy did not eliminate the transduced clone entirely.

CGD Patients with CGD have increased susceptibility to pyogenic infections caused by a defect in neutrophil oxidase activity. A defect in any of 4 proteins of the reduced nicotinamide adenine dinucleotide phosphate oxidase complex can be responsible for this disease,53 with about two thirds of the patients with CGD having a deficiency in the X-linked gene encoding gp91phox. CGD can be cured with HLA-identical allogeneic BMT,53 although haploidentical T-depleted BMT is not similarly successful54 because of toxicity from myeloablation regimens or GVHD. Gene therapy studies in mice with targeted disruptions of either gp47phox or gp91phox have been performed with demonstrable correction of oxidase activity and protection from bacterial infections.55,56 Clinical benefit in human subjects might require as little as 5% of cells with normal oxidase activity.57 After successful in vitro correction of human cells, a Phase I clinical trial was carried out in which autologous, corrected CD34+ cells from gp47phox-deficient adults

were reinfused. The study showed transient restoration of low levels of oxidase activity detectable in 0.06% to 0.2% of peripheral blood neutrophils.58 These data suggest that gene therapy protocols for CGD and other PIs in which there is no physiologic selective advantage for corrected cells will need either myeloablation, more efficient gene transduction, or both to generate a large enough proportion of corrected cells to be clinically beneficial.

Leukocyte adhesion deficiency type I Leukocyte adhesion deficiency (LAD) type I is an autosomal recessive disease caused by deficient expression of CD18, which is essential for neutrophils to migrate from the circulation to sites of inflammation. Patients with LAD have recurrent and severe bacterial infections that shorten their life expectancy.59 The only curative treatment for LAD has been BMT. A human clinical gene therapy trial was conducted for LAD by using granulocyte colony-stimulating factoremobilized peripheral blood HSCs and a retroviral vector carrying the CD18 cDNA.60 One month after infusion of transduced cells patients demonstrated that 0.03% of circulating myeloid cells contained the retrovirally transduced gene. However, these cells had no intrinsic survival advantage and were not detectable at the 2-month evaluation.60 Similar to CGD, future clinical trials might include myeloablation to increase the chances of engraftment. WAS WAS is an X-linked hematologic disorder characterized by eczema, thrombocytopenia, and dysfunction of T cells, B cells, and macrophages. WAS is caused by defects in the WAS protein (WASP) that links the cytoskeleton to intracellular signal transduction pathways.61 Severe hemorrhage, disseminated viral or other opportunistic infections, autoimmune disease, or lymphoma can cause recurrent and chronic illness and premature death. BMT from an HLA-identical sibling is the treatment of choice and can be curative for WAS, but the risks of BMT from other sources are high.62 Skewed X-inactivation in female carriers of WAS63 and the occurrence of spontaneous somatic mutations restoring WASP function associated with clinical improvement and repopulation by the wildtype phenotype64 indicate a survival advantage of genecorrected cells over WASP-deficient cells. Preclinical studies have shown that retroviral transduction corrects the immunologic defect in WASP knockout mice and in human cell lines, with increased actin polymerization and functional correction of T cells after transduction.65-68 Gene therapy clinical trials have not been performed yet. Other immunodeficiency diseases There are other PIs being investigated for potential treatment for gene therapy. Defects in Bruton tyrosine kinase (BTK) cause X-linked agammaglobulinemia (XLA), which presents with absence of B cells, low immunoglobulin levels, and frequent bacterial infections. Currently, patients with XLA receive supportive therapy with prophylactic antibiotics and IVIG; these treatments

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offer considerable protection, which is, however, expensive and incomplete.69 Mouse models of XLA have successfully been corrected with retroviral vectors carrying the BTK gene.70 Survival advantage of normal B cells over cells with BTK mutations has been demonstrated.71 XHIM1, caused by defects in the gene for CD40 ligand (CD40L), is characterized by lack of immunoglobulin switching and defects in cellular immunity because of the absence of interaction between CD40L, which should be expressed in T cells, and CD40, expressed in both T and B cells. Studies in mice exploring the possibility of gene transfer demonstrated that tightly controlled regulation of the expression of CD40L is important to avoid uncontrolled lymphoproliferation. Transgenic mice overexpressing CD40L had lymphocyte proliferation that progressed to lymphomas.72 Brown et al73 attempted gene transfer into bone marrow and thymic cells of mice lacking CD40L. Restoration of humoral and cellular immunity was detected; however, 12 of 19 mice had lymphoproliferative disorders. These studies underscore the complexity and danger of gene therapy when it is aimed at correcting highly regulated genes. Efforts to develop human gene therapy for XHIM1 will have to include effective regulatory elements of CD40L expression. Jak3-deficient SCID74,75 and RAG-deficient SCID76 are 2 other PIs for which murine models of gene therapy have shown functional correction of T and B cells. No toxicity of constitutional expression has been shown in these studies. In addition, Jak3-corrected T cells have shown a selective advantage over Jak3-deficient T cells, similar to XSCID.

INSERTIONAL MUTAGENESIS AND LEUKEMIA As discussed above, 2 patients with XSCID in the French gene therapy trial had leukemic expansions of Tcell clones 30 and 34 months after infusion. These serious adverse events led to a voluntary hold of all retroviral gene therapy clinical trials for several months and prompted investigators to reassess risks of retroviral gene transfer, particularly to hematopoietic cells. Lymphocytosis with blasts in peripheral blood, anemia, low platelet counts, and splenomegaly were found in both patients. Flow cytometric studies showed monoclonal proliferation of a single T-cell clone with a cd T-cell receptor in the first patient and 3 distinct T-cell clones with ab T-cell receptors in the second patient. Inappropriate expression of the LIM domain only-2 (LMO-2) transcription factor, caused by insertion of the IL2RG-bearing therapeutic retroviral vector close to the 59 end of the LMO2 gene locus, was found in both cases (Fig 1). LMO-2 is expressed in early hematopoietic progenitors, and around 10% of T-cell leukemias have translocations involving this locus.77 Both patients’ malignant cells also had evidence of multiple molecular events combining to cause malignant transformation. Cytogenetic translocations typical of T-cell leukemias were detected: a chromosome

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Reviews and feature articles FIG 1. Schematic representation of the LMO2 locus and the 2 retroviral insertion sites identified in the leukemic clones of patients P4 and P5 from the French XSCID gene therapy trial. Blue boxes are LMO2 exons. Green boxes linked with a black line represent the IL2RG-containing retroviral vector, with the green regions representing the retroviral long terminal repeat, a duplicated region that functions to promote expression of the IL2RG cDNA. Red arrows indicate insertion sites in clones of patients P4 and P5. Thin arrows indicate direction of transcription. Adapted from Hacein-Bey-Abina et al.25

6;13 translocation in the first patient and trisomy 10 plus a SIL-TAL1 fusion transcript in the second.25 Replicationcompetent retroviruses were not detected. Genetic and environmental influences might have contributed to the leukemia in the first case. The patient had a family history of childhood cancer and had experienced a varicella infection just before the amplified clone was discovered. Additional important potential risk factors in both children relate to their young age at treatment. These children had been given a diagnosis of XSCID on the basis of family history and were treated at 1 and 3 months of age, whereas the other infants in the trial were in the second half of their first year. Not only did the small size of the infants result in their receiving relatively large doses of gene-corrected cells (18-20 million CD34+ cc+ cells per kilogram of body weight), but also their cells were noted to proliferate more vigorously in the transduction culture than the cells of older subjects.25 HSCs obtained and gene modified from very young patients might have an intrinsically high capacity for replication, possibly increasing their risk of having a harmful gene insertion during retroviral transduction. Before 2002, retroviral insertional mutagenesis was widely assumed to be an event that occurred at random and with a very low probability of adverse consequences. Except for one study describing myeloid leukemia in mice receiving mouse bone marrow transduced with a truncated nerve growth factor receptor gene,78 animal studies in which HSCs have been transduced with replicationincompetent retroviral vectors have not shown deleterious effects attributable to the integration of the gene vector. More than 40 human clinical trials have used retroviral vectors to transfer marker genes, anti-HIV genes, genes conferring resistance to cancer chemotherapy, and genes to correct inherited disorders.78 No adverse effects from gene transfer have been observed outside of the French

XSCID gene therapy trial, even with follow-up for more than 10 years in some cases. However, most of these studies achieved only very low levels of transduction that did not persist. Several hypotheses to explain the occurrence of leukemia in the French XSCID trial and not in other similar trials have been proposed. XSCID is a condition in which gene-corrected cells have a selective advantage that overcomes the low efficiency of gene transfer. LMO-2 is a growth factor normally expressed in the earliest hematopoietic progenitor lineages. That the LMO2 locus was a target for insertional mutagenesis in both XSCID leukemia cases suggests that inappropriate LMO-2 activation in developing T cells combined with the restored cc chain can lead to excess proliferation of a Tcell progenitor, accumulation of additional oncogenic somatic changes, and ultimately leukemia. The high numbers of transduced cells in the young infants increased the chance of an integration that could result in activation of LMO-2. Previous studies suggested that retrovirus genomes have a tendency to integrate into regions of open chromatin.79 With the availability and continuing refinement of the complete human genome sequence,80 the identification of retroviral integration sites has become possible. Reports analyzing integration sites of murine retrovirus and lentivirus-based gene vectors into HeLa cells, human peripheral blood cells, and CD34+ cells have recently revealed a tendency to integrate in actively transcribed genes.81-83

CONCLUSIONS Current treatment for severe PIs is not satisfactory. Many children affected with these previously fatal

diseases are now being rescued with allogeneic BMT. Although a major gain in patient survival has been achieved with the development of BMT, many treated patients continue to have some degree of immunologic compromise, including increased susceptibility to infections, dysregulated immunity, autoimmunity, and GVHD. Gene therapy is a promising alternative treatment for single gene immunodeficiencies that can be treated with BMT, provided protein expression can be achieved in an appropriately regulated manner. Gene therapy has successfully restored immune competence in human clinical trials for XSCID and ADA-deficient SCID. However, the risk of leukemia caused by retroviral insertional mutagenesis must be understood and addressed. Although specific factors associated with patients with XSCID and cc expression might have played a role in these adverse outcomes, more clinical experience will be necessary to determine the contributions of gene defect, patient age, clinical state, and in vitro manipulations for cell transduction. The risk of oncogenesis in successful gene therapy trials will be known only after treating more patients and closely following them long term, with careful monitoring of insertion sites and clonality. While more clinical data are accumulated, new strategies are being explored, including the development of vectors that might use selfinactivating or insulator sequences to increase safety.84,85 The successes of XSCID and ADA-deficient SCID gene therapy clinical trials, as well as the unanticipated adverse events in 2 children, demonstrate that gene therapy is in its infancy but has great potential. Although further basic research is needed to learn how to make integrating gene vectors safer, clinical trials are important to develop improved treatments for all individuals with PIs. Further patient trials will be conducted with heightened monitoring and will be limited to patients who face high risks from available therapies or in whom standard treatments have failed. REFERENCES 1. Buckley RH. Primary immunodeficiency disease: dissectors of the immune system. Immunol Rev 2002;185:206-19. 2. Smith CIE, Ochs HD, Puck JM. Genetically determined immunodeficiency diseases. In: Ochs H, Smith CIE, Puck JM, eds. Primary immunodeficiency diseases, a molecular and genetic approach. New York, NY: Oxford University Press; 1999. p. 3-11. 3. Chapel H, Geha R, Rosen F, for the IUIS PID classification committee. Primary Immunodeficiency diseases: an update. Clin Exp Immunol 2003;132:9-15. 4. Cooper MD, Lanier LL, Conley ME, Puck JM. Immunodeficiency disorders. In: Hematology 2003 (Am Soc Hematol Educ Program Book). Washington, DC: American Society of Hematology; 2003. p. 314-30. 5. Buckley RH, Schiff RI, Schiff SE, et al. Human severe combined immunodeficiency: genetic, phenotypics and functional diversity in one hundred and eight infants. J Pediatr 1997;130:378-87. 6. Dadi HK, Simon AJ, Roifman CM. Effects of CD3d deficiency in maturation of a/b and c/d T-cell lineages in severe combined immunodeficiency. N Engl J Med 2003;349:1821-8. 7. Ridanpaa M, van Eenennaam H, Pelin K, et al. Mutations in the RNA component of RNase MRP cause a pleiotropic human disease, cartilagehair hypoplasia. Cell 2001;104:195-203.

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