Primary – molecular basis and gene therapy

Alain Fischer, Geneviève de Saint Basile, James P. DiSanto, Marina Cavazzana-Calvo INSERM U 429, Hôpital Necker-Enfants Malades, Université René Descartes, 149 rue de Sèvres, Paris Cedex 15, France

Introduction cations that consist not only in infections but also in allergic and autoimmune manifestations as well Primary immunodeficiencies (PID) consist in ap- as in cancer (particularly lymphomas). proximately 80 distinct diseases that disturb de- velopment and/or function of the immune system. It is estimated that about 1 in 5000 individuals is born with or will develop a symptomatic primary Classification of primary immunodeficiencies . It is usual to classify PID in four groups: i) predominant T-cell deficiency, ii) Significant advances in the molecular understand- predominant B-cell deficiency, iii) deficiencies of ing of several PID have occurred during the last the cell system and iv) complement de- three years; affected genes from 9 PID have been ficiencies. They represent 20, 70, 10 and ~1% of identified within this period of time. A list of PID the overall group of PID, respectively. Several PID for which a molecular basis is now understood is can induce life threatening and recurrent compli- given in tables I, II and III.

Table I: T-cell immunodeficiencies. Disease Phenotype Gene product X-linked SCID Absence of T and NK cells IL-2Rγ (γc) Adenosine deaminase (ADA) deficiency Reduction in T, B and NK cells ADA Purine nucleoside phosphorylase Progressively reduced counts PNP (PNP) deficiency HLA class II deficiency (group A) Low CD4 T-cell counts, defective ClassIItrans-activator response (CIITA) TAP 2 deficiency Reduced class I expression, low NK activity TAP 2 (peptide transporter) ZAP 70 deficiency Low CD8 T-cell counts, defective activation ZAP 70 tyrosine kinase of CD4 T-cells CD3 ε deficiency Defective T-cell activation CD3 ε CD3 γ deficiency Low CD8 T-cell counts, defective T-cell activation CD3 γ Wiskott-Aldrich syndrome (WAS) Progressive lymphocytopenia, defective antibody WASP (nuclear protein) response to polysaccharides, thrombocytopenia “Hyper IgM syndrome” Defective Ig heavy chain switch, defective handling CD40 ligand of mucosal opportunistic microorganisms

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T-cell immunodeficiencies mRNA translation [8]. Surprisingly, some residual T-cell differentiation occurs as observed in mice The most severe form of PID consists in severe in which the c gene has been inactivated [9], sug- combined immunodeficiencies (SCID) charac- γ terised by a profound block in T-cell differentia- gesting a partially effective supplementary path- tion and thus by a virtual absence of mature T- way for proliferation of early T-cell precursors. cells. SCID are leading to severe infections occurring in the first year of life causing death in Adenosine deaminase deficiency the absence of treatment by allogeneic bone mar- Adenosine deaminase (ADA) deficiency was the row transplantation. Frequency is estimated to be first identified PID. Accumulation of deoxyadeno- about 1 in 75 000 live births [1]. sine results in excessive concentration of deoxy- ATP in precursors. The latter product X-linked SCID blocks DNA synthesis by inhibiting synthesis of Two molecular defects have been identified that the other deoxynucleotides. The consequences of can cause SCID in human beings. The most fre- this block are limited to because quent form of SCID, i.e. X-linked SCID (about 50 5’nucleotidase reduces deoxy-ATP level in other to 60% of cases of SCID), is characterised by an cell types [10]. The residual ADA enzymatic ac- absence of mature T- and natural killer (NK) cells tivity determines PID severity, i.e., between full while B-cells are present in normal or elevated blown expression (alymphocytosis) to a mild im- numbers. The thymus is profoundly depleted of munodeficiency with late onset symptoms. A thymocytes. The X-linked SCID locus was as- residual activity of 10% of normal ADA activity is signed to Xq1.3-2.1. It was then recognised that sufficient to avoid any expression of immunodefi- the gene encoding the γ chain of the interleukin-2 ciency. ADA deficiency can be treated by weekly (IL-2) receptor, renamed γc, maps to the same re- intramuscular injection of bovine ADA coupled to gion of the X chromosome than the X-linked SCID polyethyleneglycol (PEG) or by bone marrow locus and was found mutated in patients with X- transplant. PEG-ADA is quite stable in vivo. The linked SCID [2]. A variety of mutations affecting enzyme is active and enables some T- and B-cell all parts of the gene has now been described [3, 4]. differentiation and function. About 40 patients Interestingly, it is not primarily because the high have been treated world-wide for a maximum fol- affinity IL-2 receptor (IL-2R) is lacking as a conse- low-up of 8 years. The treatment’s efficacy varies quence of the absence of the γc chain that T-cell between patients and although PEG-ADA treat- differentiation is blocked. Indeed, PID patients ment does not achieve full correction of the im- with selective IL-2 deficiency, or mice unable to munodeficiency it appears to be more effective in make IL-2 because the IL-2 gene has been inacti- milder forms of the ADA deficiency. This treat- vated, have mature T-cells indicating that T-cell ment nonetheless allows most patients to survive differentiation can occur in the absence of IL-2/ in good condition and is rather safe until a cura- IL-2R interaction. The γc chain has been found to tive therapy becomes available. be a member of several receptors, i.e. Other forms of SCID, mainly characterised by IL-2, IL-4, IL-7, IL-9 and IL-15 [5]. The SCID phe- both defective T- and B-cell differentiation, have notype thus results from the summation of these been described. They possibly correspond to de- defects among which the IL-7 receptor anomaly fects in the machinery necessary for T-cell recep- may play a major role in blocking early T (NK)- tor (TCR) and immunoglobulin gene rearrange- cell precursor proliferation. ments as observed in the murine SCID model; Attenuated phenotypes have been described. among the defects studied are those in rag-1 and They are characterised by oligoclonal T-cell dif- rag-2 proteins necessary for the initiation of the ferentiation, found in patients with either reduced recombination process [11]. γc expression [6] or with mutation of γc in the binding site of the Jak3 tyrosine kinase [7]. An X- Atypical SCID linked SCID has been described in a dog pedigree; A number of inherited T-cell immunodeficiencies, the γc chain is deficient because of an early stop in sometimes called “atypical SCID” or “combined

26 Annales Nestlé 1996;54:25-34 Primary immunodeficiencies – molecular basis and gene therapy immunodeficiencies” (CID), remain ill defined 50% of normal, respectively) and partially defec- although some T-cell differentiation occurs. tive signal transduction [14, 15]. These deficits are characterised by defective T- cell activation or effector function, although some ZAP deficiency T-cell differentiation does occur. Very interesting Recognition by the TCR of peptides associated findings were recently reported which led to the with HLA molecules induces a cascade of signals, molecular characterisation of diseases described the first of which involves a series of tyrosine ki- below. nases: lck, fyn and ZAP 70. A ZAP 70 deficiency has been demonstrated in several immunodefi- HLA class II deficiency cient patients. Absence of detectable ZAP 70 acti- Some patients present with the defective expres- vity blocks signal transduction in T-cells empha- sion of HLA class II molecules caused by the ab- sizing its crucial role in the initiation of T-cell normal transcription of otherwise structurally nor- activation. It also leads to a lack of CD8 T-cells mal genes. Defective expression of HLA class II presumably because ZAP 70 cannot be replaced molecules leads to a reduction (but not a disap- satisfactorily by a similar kinase, syk, in thymo- pearance) of the CD4+ T-cell pool as well as lack cytes following class I (CD8 dependent) restricted of HLA class II restricted T-cell responses and activation [16, 17]. defective antibody responses. Complementation analysis revealed three distinct groups of HLA Wiskott-Aldrich syndrome class II deficiency. In two, the promoter region of The Wiskott-Aldrich syndrome (WAS) is charac- HLA class II genes is not associated with DNA terized by the association of complex features, binding proteins in intact cells, thus the DNA bind- i.e. progressive T- and B-cell deficiency with de- ing proteins that interact with the so-called X1 fective antibody responses to polysaccharides, box of the promoter may be missing. In the third allergic and autoimmune manifestations, and (group A), the promoter is normally occupied; but quantitative and qualitative (small volume) a “class II transactivator” (CIITA) nuclear protein anomalies. The WAS locus was assigned is defective in these patients. The gene was cloned to Xp11.23. The WAS gene was identified by by complementation. Its product, which is γ- in- positional cloning. It encodes a nuclear protein of terferon inducible, is necessary for class II expres- unknown function that is expressed by lympho- sion. Patients from group A exhibit mutations of cytes and [18]. the CIITA encoding gene [12]. “Hyper IgM syndrome” TAP 2 deficiency The hyper IgM syndrome (HIGM-1) is primarily a TAP 2 is required as an heterodimer with TAP 1 to B-cell deficiency. It is characterized by a defec- transport peptides from the cytosol to the endo- tive Ig switch to IgG, IgA and IgE, but IgM and IgD plasmic reticulum, where they interact with HLA are produced normally. It is most frequently X- class I molecules. Recently a deficiency of TAP 2 linked. B-cells from X-linked HIGM patients pro- has been described in two siblings. As expected, it duce IgG, IgA and IgE stimulated with an anti- resulted in low CD8 T-cell counts but surprisingly CD40 antibody and the necessary (IL-4, also to reduced NK activity [13]. IL-10,...). This observation suggests that the disease may be the consequence of a deficiency in CD3 γ and ε deficiencies the CD40 ligand, a molecule expressed by activa- Some PID are caused by a block in the signal ted T-cells. Indeed, the disease locus maps to Xq transduction which occurs after presenta- 26, where the CD40 ligand encoding gene was as- tion to T cells and normally lead to T cell activa- signed. Activated T-cells of patients with HIGM-1 tion. In two families, deficiencies in respectively do not express functional CD40 ligand and carry CD3ε and γ subunits of the TCR/CD3 complex various mutations of the CD40 ligand gene [19, have been found leading to reduced expression of 20]. This finding shed light on the dual mecha- the TCR/CD3 complex on the membrane (10 and nism of the Ig switch, i.e., that it requires both

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CD40 ligand/CD40 interaction and cytokine (IL-4, tized micro-organisms by and IL-10,..) signalling. The HIGM-1 syndrome, how- . The oxidative burst resulting in the ever, also frequently causes neutropenia. Oppor- superoxide anion and other oxygen derivatives tunistic infections caused by Pneumocystis carinii production involved in microorganism killing is and Cryptosporidia are common in these patients. not occurring in this setting. Four different genetic Both defects are likely accounted for by the ex- defects have been described. They correspond to pression of CD40 by haematopoietic precursors, abnormalities in four proteins involved in the spe- macrophages and/or epithelial cells, respectively. cific electron transport chain required to produce Therefore, multiple interactions between activa- O2°, the NADPH oxidase. The NADPH oxidase ted T-cells and various cell types play important complex is composed by two subunits of cy- roles in defence against infections. tochrome b (p91 and p22) and two proteins loca- lized in the cytosol (phox 47 and 67), which B-cell immunodeficiencies translocate to the membrane following cell activa- tion. The gene encoding p91 is located on the X-linked agammaglobulinemia (XLA) was the short arm of the X chromosome. Mutations of p91 first PID to be described. XLA is characterized by cause more than half of CGD cases. The other low to absent serum immunoglobulins and mature three genes are located on autosomes; CGD are B-cells are barely detectable. In contrast, pro- and thus inherited in an autosomal recessive manner pre-B cells are present in the marrow and T-cells [25] (Table III) . are normal. Following gene locus mapping to Xq 22.1-22.2, the XLA related gene was identified by Table III: Phagocyte cell immunodeficiencies. positional cloning [21-23]. It encodes for the Bru- Disease Phenotype Gene product ton tyrosine kinase (btk), which is partly analo- gous to the src tyrosine kinases and is expressed Chronic Defective p91 phox all along the B-cell developmental pathway. It granulomatous oxidative burst disease (CGD) u cyto- contains several domains which interact with X-linked chrome b other proteins: the pleckstrin, a SH2- and a SH3, CGD A-R1 idem p22 phox and the kinase domains. The btk is not involved in Ig gene rearrangement since it occurs in its ab- CGD A-R idem p47 phox sence, but may be important for further cell acti- CGD A-R idem p67 phox Leukocyte adhesion Defect in adhesion β2 integrin vation. Mice with the X-linked immunodeficiency deficiency (LAD) to endothelial cells (CD18) (xid), i.e. a defect in thymo-independent antigen and chemotaxis type 2 antibody responses, also carry a mutation in the btk encoding gene [24] (Table II). 1 A-R autosomal recessive Table II: B-cell immunodeficiencies. Leukocyte adhesion deficiency LAD Disease Phenotype Gene product Leukocyte adhesion deficiency ( ) is charac- terized by the accumulation of granulocytes in the X-linked agamma- A(hypo)gamma- btk tyrosine- blood stream. Therefore, they do not migrate to globulinemia globulinemia kinase sites of infection. Granulocytes and IgG1 deficiency IgG1 deficiency γ1 deletion bind poorly to endothelium and fail to migrate κ chain deficiency Absence of Igκκ through them. This defect is caused by an abnor- mal leukocyte expression of β2 integrins, i.e. LFA-1, Mac-1 and p150-95 [26]. These three hete- Phagocyte cell immunodeficiencies rodimeric proteins share a common β2 subunit but the α-subunits of these proteins are distinct. Chronic granulomatous diseases All three proteins are involved in cell adhesion Chronic granulomatous diseases (CGD) are cha- due to their ability to bind intercellular adhesion racterized by a default in the killing of phagocy- molecules (ICAMs).

28 Annales Nestlé 1996;54:25-34 Primary immunodeficiencies – molecular basis and gene therapy

Treatment of primary immunodeficiencies Other strategies however exist to increase the fraction of dividing stem cells, either their in vitro Life threatening immunodeficiencies, mainly T- incubation with cytokines (various cocktails of cell immunodeficiencies, require allogeneic bone IL-1, IL-3, IL-6, SCF, GM-CSF and LIF have been marrow transplantation (BMT) as curative treat- tested) or by the in vivo pre-treatment with drugs ment. Results are excellent when an HLA identical such as 5 fluoro-uracile that kill dividing haema- donor is available. Approximately 90% of patients topoietic cells and leads to secondary-induced are cured by this procedure. When such donors are mobilization of the stem cell pool. The best results unavailable, the probability of a cure is between obtained so far in vivo were achieved in leukemic 40 to 60% depending on patients’ status and patients who received chemotherapy in the weeks disease severity and the degree of compatibility prior to autologous marrow harvesting and trans- between the donor and recipient. Therapeutic fai- duction; in those patients, the marker gene (and lures may be due to graft rejection, graft-versus- product) could be detected in a significant fraction host-disease or infections caused by the long- of peripheral leukocytes more than one year after lasting immunodeficiencies that follow the T-cell the autologous marrow gene transfer [28]. depleted BMT [27]. Efforts are presently being Aggressive chemotherapy prior to gene transfer pursued to develop new therapeutic strategy such is however seldom possible because of these as gene therapy. patients’ clinical status. If required, such chemo- therapy provides little clinical advantage com- pared to the reference treatment, i.e., allogeneic General considerations on gene therapy for PID bone marrow transplant. It is hoped however that gene transfer may provide a selective advantage to Gene therapy can be considered only after the re- transduced cells in settings characterized by faulty lated gene is cloned and its function and pattern of cell proliferation (such as in ADA expression are well described. Promoter and en- or γc deficien- hancer regions should be known and in some in- cy). If this hypothesis is correct, one may infer stance may be used to activate appropriate gene that a very low rate of stem cell transduction may expression. Difficulties can vary enormously. For ultimately lead to a cure. X chromosome inactiva- tion patterns in obligate carriers of γc(-) X-SCID fit instance, on one hand ADA, which is an ubiqui- with this model since all lymphocytes, present in tously expressed house keeping gene and, on the normal numbers, stem from cells in which the other hand CIITA, the physiological expression of “healthy” X chromosome is active. The hypothe- which being under strict regulation by γ interferon. sis however has not yet been tested because of the lack of a suitable animal model. Bone marrow gene therapy Two main strategies can be considered for gene Leukocyte gene therapy therapy to correct PID in humans: (i) targeting Targeting mature lymphocytes or monocytes is haematopoietic stem cells with the aim of a defini- technically easier because the former can be in- tive cure or (ii) targeting peripheral leukocytes duced to proliferate in vitro and to therefore inte- with the aim of gene expression over a limited grate and express a transgene provided by a retro- time. The first goal is very complex. Most pluri- viral vector. However, the life span of activated potent haematopoietic stem cells are in G0/G1 and T-cells is limited to weeks in vivo. It is also un- thus inaccessible to vectors such as retroviruses clear whether such cells return to secondary lym- which commonly provide gene integration into phoid organs and thus reconstitute a large size im- the genome of dividing cells. They are, however, mune repertoire. In any case, use of this strategy theoretically accessible to other vectors like will require repeated leukocyte apheresis, a cum- adenoviruses which can persist as episomes in bersome procedure. Transduction of monocytes non-dividing cells. However, once cells divide as also can be achieved with several vectors. Once any haematopoietic progenitor cell eventually reinfused, however, life span will then be limited does, the episome will not be replicated. to months.

Annales Nestlé 1996;54:25-34 29 Alain Fischer, Geneviève de Saint Basile, James P. DiSanto, Marina Cavazzana-Calvo

Level of expression and safety issues Transduced peripheral T-cells can proliferate nor- It is important to achieve levels of gene expression mally and exert cytolytic activities in vitro [31]. in transduced cells which are sufficient for func- Ferrari et al. have transferred transduced T-cells tion and are non-toxic. For example, in γc deficien- from ADA (-) patients into immunodeficient mice cy, a residual 20% expression still cause a severe/ and recovered human cells from murine spleen; lethal immunodeficiency [6]. In other instances, they were able to derive antigen-specific T-cell persistant mutated gene products may exert a dom- lines showing in vivo and in vitro functionality of inant negative effect that precludes the efficacy of transduced cells [32]. gene transfer. This is of concern particularly for multimeric molecules such as the CD40 ligand. Clinical results Another key aspect of gene transfer deals with These positive results led the group of Blaese to safety issues. Retroviral vectors are non-toxic, un- propose a clinical trial of repeated infusions of pe- less replication-competent viruses contaminate ripheral T-cells transduced ex vivo with a retroviral the defective vector as such cases observed in vector containing the ADA cDNA. Two patients experiments performed in monkeys lymphomas (under PEG-ADA) have undergone this experimen- developed [29]. These observations underline the tal treatment. Apheresis were repeated every 4 to need for careful monitoring of replication-compe- 12 weeks. T-cells were activated by anti-CD3 anti- tent virus in gene transfer experiments. Insertional body and IL-2 for one day, then cocultured with mutagenesis leading to activation of an oncogene retroviral vector containing supernatants. One to is a theoretically possible complication of gene five percent (1-5%) of lymphocytes were trans- transfer therapies that use retroviruses. This likeli- duced, expanded in vitro for 11 days, and reinfused hood, however, appears doubtful since this possi- to the patients. No adverse effects have been noted bility has not yet been reported in experimental or in the two patients. Significant accumulation of clinical studies. ADA (+) T-lymphocytes was observed in the blood and improved immune function was observed in one patient. Reconstitution of immune function Gene therapy for ADA deficiency however was difficult to evaluate since patients ADA deficiency has been the major target of gene were also maintained on PEG-ADA treatment. transfer. Several reasons have led to this priority. Transduced lymphocytes were detected in the ADA deficiency is lethal if untreated and curable blood more than 18 months after the last injection by allogeneic BMT. Thus, it is expected that mar- of autologous lymphocytes. Although these trials row cell transduction would work. The gene re- did not demonstrate a cure, they showed the feasi- sponsible for this defect was cloned 10 years ago bility of gene transfer in humans, absence of ad- and is expressed ubiquitously and is not under con- verse effects, and gave new information on the life trolled regulation. ADA is indeed expressed as a span of transduced lymphocytes [33]. “house keeping” gene and its expression is likely to confer a selective advantage to transduced cells. Transduction of marrow cells Transduction of marrow cells by the introduction Experimental results of a normal ADA gene was achieved in vitro, usu- Retroviral vectors, usually derived from the ally in immature cells expressing the CD34 Moloney retrovirus, have been used for ADA gene molecule. It has been possible to achieve integra- transfer experiments. Several groups have demon- tion and expression of ADA in long-term cultures strated that efficient transduction is achieved ex initiating cells (LTC-IC) for more than 5 to 7 weeks vivo in peripheral ADA (-) lymphocytes, i.e. acti- [34]. These observations document that the effi- vated T-lymphocytes or EBV-transformed B-lym- cient transduction of highly immature human phocytes. Expression is stable owing to ADA gene marrow cells is possible but do not demonstrate integration into the genome and sufficiently effec- transduction of pluripotent haematopoietic cells tive to correct pre-existing metabolic defects, i.e., with the potential for self renewal. Nonetheless, the clearance of 2’ deoxyadenosine [30, 31]. these results achieved under conditions approved

30 Annales Nestlé 1996;54:25-34 Primary immunodeficiencies – molecular basis and gene therapy for clinical study (e.g. the use of supernatants con- Myeloablative treatment was not provided to taining high titer of retroviral vectors) are quite the patients who also received PEG-ADA initiated encouraging. prior to or soon after gene transfer. Two patients Although efficient transduction can be achieved received both repeated injections of transduced in vivo in murine models in which transduced unseparated marrow cells and lymphocytes (cells haematopoietic stem cells provide long-term ex- were transduced by two different vectors). No pression of human ADA (even after the secondary side effects have been noted in these patients. The and tertiary transplantation syngeneic mice [35- integration in marrow and peripheral leukocytes 38]), gene expression is stable and high enough of the transferred gene was detected within the for potential therapeutic applications. Efficient first months following treatment, but evidence of transfer into murine haematopoietic stem cells a progressive decline with persistent low levels at likely is explained by the murine origin of the one year have been noted with longer follow-ups. retroviral vectors used in those experiments. Re- Because only a relatively low number of CD34 sults, however, are less impressive in large ani- cells were infused and transduced (5 to 12% of mals. In monkeys, following lethal irradiation, / progenitor cells in a cli- autologous infusion of transduced marrow cells nical trial performed in Paris and London in col- led to long-term detection of the human ADA gene laboration with D. Valerio, Leiden), there is sig- in only 1 per 1000 peripheral granulocytes and nificant room for improvement in future studies. lymphocytes; these results indicate a low rate None of the patients, however, have been cured transduction of monkey stem cells [39]. Slightly by gene transfer. Transduction of a sufficient better results were achieved more recently in number of haematopoietic stem cells to effect a monkeys by using the following protocol: (i) permanent cure could thus be very difficult to monkeys were treated first with 5 fluoro-uracile to achieve without inducing more stem-transduced induce stem cells to divide; (ii) five days later, cells to divide. Alternatively, use of the adeno- marrow cells were harvested, infected by a retro- associated virus (AAV) vector could achieve a viral vector, cultured on a stromal cell line ex- higher efficiency of transduction [41]. This de- pressing stem cell factor and reinjected after 2 pends, in part, on reproducibly obtaining high doses of 6.5 gray irradiation of the monkeys. This viral titers free of adenovirus. A similar in vitro procedure led to the long-term detection of human strategy has shown that the PNP gene can be ADA in 1-2% of granulocytes and to lymphocytes expressed in PNP (-) lymphocytes [42]. with a 3% human ADA activity compared to simian ADA levels [40]. Although these experiments achieved long-term X-linked SCID-γc deficiency transduction with low efficacy, they nevertheless Due to the severity of the disease, X-linked SCID- indicated that infection of haematopoietic stem γc deficiency is also a good candidate for gene cells can be achieved in large mammals with transfer. The γc gene is expressed early during retroviral vectors. Owing to the lack of appro- lymphocytic and myeloid differentiation and by priate ADA deficient animal models, it is not pos- mature lymphocytes. As is true for ADA deficien- sible to assess whether such levels of transduction cy, one expects a selection advantage in early lym- will be sufficient to cure patients with ADA defi- phocyte progenitors capable of γc expression (see ciency. The putative selection advantage of trans- above). The recent creation of a γc(-) mouse offers duced cells recently led to the treatment of several the possibility to test in an animal model putative patients with autologous haematopoietic cells selection advantage of transduced cells and the either from marrow or cord blood origin that were possible toxicity of an inappropriate expression of infected ex vivo with retroviral vectors containing γc by cells of different haematopoietic lineages. the ADA cDNA. The latter transgene was placed Since the γc gene is short (4.2 kb), full integration either under viral long terminal repeat (LTR) tran- with its promoter into retroviral constructs is possi- scription control (modified or not) or under inter- ble. Thus, its physiological regulation may be nal promoter control. achieved. By using Moloney derived retroviruses,

Annales Nestlé 1996;54:25-34 31 Alain Fischer, Geneviève de Saint Basile, James P. DiSanto, Marina Cavazzana-Calvo we have been able to achieve expression of γc in hesion-capability [46]. Long-term expression of EBV transformed γc (-) human B-cells and gene in- human CD18 also was achieved in mice following tegration into LTC-IC (up to 7 weeks) from CD34+ transfer into marrow cells [47]. The existence of a marrow cells. More work in this form of SCID is CD18-low murine strain obtained by homologous needed to optimize transfer conditions which recombination and of CD18 (-) cattle offers very would be applicable in clinical trials. Questions interesting models to assess diverse experimental which remain to be answered are very similar to issues raised by the possibility of gene transfer as those raised for gene therapy of ADA deficiency. a cure for leukocyte adhesion deficiency.

Chronic granulomatous diseases Conclusion Correction of three of the genetic defects causing chronic granulomatous diseases (CGD) also has Although there is much work to be done before been achieved by gene transfer in EBV B-cells. gene therapies provide a cure for patients with Retroviral vectors and more recently AAV vectors severe PID, advances have occurred rapidly in the have been used in those investigations. Expres- last years. The identification of affected genes and sion of p91 phox, p22 phox and p47 phox has the initiation of clinical trials lead us to anticipate been induced leading to 02° production levels much progress in a few years. Several lines of re- reaching 7 to 30% of normal values [43-45], a de- search appear particularly promising: (i) the de- gree of correction thought to be sufficient to avoid sign of more efficient retroviral vectors [48] and signs and symptoms associated with these di- the effective use of AAV-derived vectors to pro- seases. The p91 phox and p22 phox genes also vide high efficiency transduction [41]; (ii) better have been transferred in p91 phox (-) and p22 approaches for selecting haematopoietic stem phox (-) CD34 marrow cells, respectively, leading cells and inducing their replication [49]; (iii) im- to 02° production at level 2.5 to 5% of normal in provement in conditions of haematopoietic stem granulocytes generated after 15 days in vitro [45]. cell infection [50]; (iv) use of long-term in vitro Although encouraging, these results are still insuf- culture in infectious conditions as shown with dog ficient (in duration and magnitude of expression) haematopoietic stem cells [51]. to achieve a cure. In contrast to gene transfer strategies to correct SCID, no selective advantage will be conferred to transduced myeloid cells and References it is likely that chemotherapy will be necessary to achieve acceptable efficacy. Therefore, another 1. Fischer A. Severe combined immunodeficiencies. Im- munodefic Rev 1992;3:83-100. strategy under consideration is gene transfer in 2. Noguchi M, Yi H, Rosenblatt HM, et al. Interleukine-2 monocytes. Monocytes are progenitors of macro- receptor γ chain mutation results in X-linked severe com- phages and can deliver an efficient oxidative bined immunodeficiency in humans. Cell 1993;73:147- burst. Injection of autologous transduced mono- 57. cytes, therefore, may help control a life-threate- 3. Puck JM, Deschenes SM, Porter JC, et al. The in- terleukin-2 receptor γ chain maps to Xq13.1 and is mu- ning infection in CGD patients. Gene transfer in tated in X-linked severe combined immunodeficiency, monocytes can be very efficiently achieved by SCIDX1. Hum Mol Genet 1993;2:1099-104. adenoviral vectors. We do not know, however, if 4. Markiewicz S, Subtil A, Dautry-Varsat A, et al. Detec- stable and adequate levels of expression can be tion of three nonsense mutations and one missense muta- tion in the interleukin-2 receptor g chain gene in SCIDX1 achieved by this approach. which differently affect the mRNA processing. Ge- nomics 1994;21:291-3. 5. Leonard WJ, Noguchi M, Russell SM, et al. The molecu- Leukocyte adhesion deficiency lar basis of X-linked severe combined immunodeficien- cy: the role of the interleukin-2 receptor γ chain as a com- The β2 integrin gene was transduced efficiently mon γ chain, γc. Immunol Rev 1994;138:61-86. into CD18 (-) EBV B-cells by using a retroviral 6. DiSanto JP, Rieux-Laucat F, Dautry-Varsat A, et al. De- vector. Gene transfer led to the restoration of ad- fective human interleukin-2 receptor (IL-2R) γ chain in

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