Background X-linked severe combined immunodeficiency due toa mutation in the gene encoding the common (c) chain is a lethalcondition that can be cured by allogeneic stem-cell transplantation.We investigated whether infusion of autologous hematopoieticstem cells that had been transduced in vitro with the c genecan restore the immune system in patients with severe combinedimmunodeficiency.
Methods CD34+ bone marrow cells from five boys with X-linkedsevere combined immunodeficiency were transduced ex vivo withthe use of a defective retroviral vector. Integration and expressionof the c transgene and development of lymphocyte subgroups andtheir functions were sequentially analyzed over a period ofup to 2.5 years after gene transfer.
Results No adverse effects resulted from the procedure. TransducedT cells and natural killer cells appeared in the blood of fourof the five patients within four months. The numbers and phenotypesof T cells, the repertoire of T-cell receptors, and the in vitroproliferative responses of T cells to several antigens afterimmunization were nearly normal up to two years after treatment.Thymopoiesis was documented by the presence of naive T cellsand T-cell antigen-receptor episomes and the development ofa normal-sized thymus gland. The frequency of transduced B cellswas low, but serum immunoglobulin levels and antibody productionafter immunization were sufficient to avoid the need for intravenousimmunoglobulin. Correction of the immunodeficiency eradicatedestablished infections and allowed patients to have a normallife.
Conclusions Ex vivo gene therapy with c can safely correct theimmune deficiency of patients with X-linked severe combinedimmunodeficiency.
Deficiency of the common (c) chain, an X-linked disorder, causesthe most frequent form of severe combined immunodeficiency disease.1,2The c chain is an essential component of five cytokine receptors,all of which are necessary for the development of T cells andnatural killer cells. Without the c chain, there is a completeabsence of mature T and natural killer cells, whereas B cellsare usually present in normal or increased numbers. Severe combinedimmunodeficiency is fatal during the first year of life becauseof severe, recurrent infections, unless transplantation of hematopoieticstem cells restores T-cell function.3,4 The survival rate aftertransplantation of HLA-identical hematopoietic stem cells ismore than 90 percent, whereas with haploidentical stem cellsit is 70 to 78 percent.3,4 In most patients, deficient B-cellfunction persists after transplantation and requires lifelongimmune-globulin–replacement therapy.3,5 Some patientsalso have persistent deficiencies of T-cell function after stem-celltransplantation.4,6 Assessment of an alternative therapy basedon the ex vivo transfer of the c gene into autologous hematopoieticprecursor cells was therefore warranted. In a preliminary report,we showed that this approach corrected the T-cell deficiencyin two patients with X-linked severe combined immunodeficiencywho were followed for 10 months after gene transfer.7 We nowreport the effectiveness of the procedure in five patients witha follow-up of up to 30 months.
Methods
Patients
Five consecutive patients without HLA-identical donors wereenrolled in the trial between March 1999 and February 2000.The main characteristics of these boys at the time of diagnosisare shown in Table 1. The diagnosis of X-linked severe combinedimmunodeficiency was based on peripheral-blood lymphocyte countsand confirmed by c mutation analysis. The protocol was approvedby the French Drug Agency and the local ethics committee, andwritten informed consent was obtained from the parents, whowere told that an alternative treatment (bone marrow transplantation)was available. All of the patients were kept in sterile isolationand received nonabsorbable antibiotics and intravenous immuneglobulin. Additional information about the five patients isavailable as Supplementary Appendix 1 with the full text ofthis article at http://www.nejm.org.
The vector containing the c chain was derived from a defectiveMoloney murine leukemia virus and has been previously described.7With the patients under general anesthesia, 30 to 150 ml ofbone marrow was obtained, and CD34+ cells in the marrow wereselected for, as described below. These cells were stimulatedto grow in X-vivo 10 medium (BioWhittaker, Walkersville, Md.)containing 4 percent fetal-calf serum (StemCell Technologies,Vancouver, B.C., Canada), 300 ng of stem-cell factor per milliliter(Amgen, Thousand Oaks, Calif.), 300 ng of Flt-3 ligand per milliliter(Immunex, Seattle), 60 ng of interleukin-3 per milliliter (Novartis,Rueil-Malmaison, France), and 100 ng of polyethylene glycol–conjugatedmegakaryocyte growth and differentiation factor per milliliter(Amgen). The cells were then transduced with a supernatant ofthe cultured c-containing vector in the presence of the precedingcytokines and 4 ng of protamine sulfate per milliliter (ChoaySanofi, Gentilly, France). The procedure was carried out insterile bags (Nexell Therapeutics, Irvine, Calif.) that werecoated with 50 ng of human recombinant fibronectin per milliliter(Takara Shuzo, Shiga, Japan). The supernatant was replaced every24 hours during the three-day transduction period. The numberof cultured cells was increased by a factor of five to eight,and 14 million to 38 million CD34+ cells per kilogram of bodyweight were infused into the patients without preparative conditioning(Table 1).
Analysis of Immune Reconstitution
Immunofluorescence analysis, assays for proliferation of peripheral-bloodmononuclear cells, analysis of the T-cell–receptor repertoire,and studies of natural-killer-cell cytotoxicity were performedas previously described.7,8,9 The presence of serum antibodiesagainst polioviruses, tetanus and diphtheria toxoids, Haemophilusinfluenzae, and Streptococcus pneumoniae was determined by enzyme-linkedimmunosorbent assays. Levels of isohemagglutinins were measuredby a hemagglutination assay. Antibody levels were determinedone to three months after three immunizations had been administered.The interval between the last intravenous infusion of immuneglobulin and the determination of antibody levels was at leastthree months.
Leukocyte Subgroups and Purification of CD34+ Cells
Peripheral-blood samples were separated into mononuclear cellsand granulocytes by centrifugation and sorted by flow cytometry(FACS Vantage, Becton Dickinson Immunocytometry Systems, SanJose, Calif.). Isolation of CD34+ progenitor cells was performedby an immunomagnetic procedure (Miltenyi Biotec, Bergisch Gladbach,Germany). Two successive immunomagnetic procedures increasedthe purity of the CD34+ population to 99 percent.
Quantification of Transgene Integration
Genomic DNA was extracted from peripheral-blood mononuclearcells and amplified with use of quantitative polymerase chainreaction (PCR). Amplification, data acquisition, and analysiswere performed with the use of a sequence detector (ABI PRISM7700, Perkin Elmer, Norwalk, Conn.). Two sets of primers andprobes were used in each PCR reaction. For the quantificationof integrated transgene sequences, the primers positioned inthe long terminal repeat and probe were as previously described.10The standard curve used as a reference for quantification ofthe viral copy number was based on serial dilutions of a plasmidranging from 40 to 4 million copies. This plasmid containedtwo copies of the long terminal repeat and one of the humanalbumin sequence (Genethon III Laboratory, Evry, France).
To define the detection limit and linear range of duplex PCR,we used a standard curve consisting of a log-scale dilutionof cells from an Epstein–Barr virus (EBV)–transformedB-cell line derived from a patient with X-linked severe combinedimmunodeficiency and containing approximately two copies ofc provirus per cell with uninfected cells from the same EBV-transformedB cell line. The lower limit of sensitivity of the method was0.01 percent of c-positive cells.
Quantification of T-Cell Antigen-Receptor Episomes
Analysis of T-cell antigen-receptor episomes in peripheral-bloodmononuclear cells was performed by real-time quantitative PCRby means of the 5' nuclease assay (TaqMan) with an ABI PRISM7700 system (Perkin Elmer).11,12 PCR conditions as well as primersand probe sequences are available on request.
Presence of Integrated Provirus after Long-Term Culture of CD34+ Cells
Purified CD34+ cells were cultured for six weeks on irradiatedMS-5 stromal feeder layers in a limiting-dilution assay (10,000to 150 cells per well) as described previously.13 After sixweeks, the cells were assayed for colony-forming units. Subsequently,for each dilution, all colony-forming units obtained on day14 from the same dish were pooled. DNA was analyzed by PCR todetermine the percentage of c-positive dishes.
Results
Clinical Outcome
After infusion of CD34+ cells that had been transduced in vitrowith the c gene, four of the five patients (Patients 1, 2, 4,and 5) had a clear-cut clinical improvement (Table 1). Pulmonaryinfections in Patient 1 and Patient 2 cleared and did not recur,and graft-versus-host–like skin lesions, a feature ofsevere combined immunodeficiency, disappeared in Patient 2 andPatient 5 within the first 50 days after gene therapy. Patient1 and Patient 2 left the sterile environment on day 90, andPatient 4 and Patient 5 left on day 45. In Patient 1 and Patient2, protracted diarrhea resolved, and parenteral nutrition wasdiscontinued four months and three months after gene therapy,respectively. None of these four patients have subsequentlyhad severe infections. Intravenous immune globulin was discontinuedthree to four months after gene therapy. Growth and psychomotordevelopment have been normal to date. Patients 1, 2, 4, and5 are now living at home in normal environmental conditions.
Patient 3, in whom reconstitution of T cells failed, underwentsplenectomy four months after gene therapy for persistent splenomegalycaused by a disseminated bacille Calmette–Guérininfection. A rescue stem-cell transplantation from an unrelateddonor matched at HLA-A, B, DR, and DQ loci but mismatched atone HLA-C locus was performed after eight months, accordingto the protocol. At the last follow-up visit, partial T-cellimmunity had been restored in this patient.
T-Cell Development
In Patients 1, 2, and 4, the number of T cells increased progressivelyand reached normal values for age three to four months aftergene therapy; they were within the normal range at the lastfollow-up visit (Figure 1). In Patient 5, the initially highnumber of maternal T cells (Table 1) disappeared within threemonths after treatment, while autologous T cells appeared.
Figure 1. Absolute Numbers of CD3+ Cells after Gene Transfer in Patients 1 through 5.
Quantitative analysis of provirus integration indicated that100 percent of the T cells from Patients 1, 2, 4, and 5 containedthe transgene (Figure 2). On Southern blotting, there were oneto three provirus integration sites per cell (data not shown).All T cells in Patients 2, 4, and 5 expressed cell-surface receptorswith the c chain. In all four patients, there was a normal distributionof T cells with / or / receptors, and the numbers of CD4+ andCD8+ T cells were similar to those in age-matched controls (datanot shown). Conversely, no T cells were detected in the bloodof Patient 3 up to six months after treatment (Figure 1).
Figure 2. Frequency of Sorted T Cells (CD3+), B Cells (CD19+), Monocytes (CD14+), and Granulocytes (CD15+) Containing the Common (c) Chain after Gene Therapy in Patients 1, 2, 4, and 5.
Real-time quantitative polymerase-chain-reaction analysis of DNA was used to determine the frequency of vector-containing cells, as described in the Methods section.
Analysis of naive (CD45RA+) and memory (CD45RO+) subgroups withinCD4+ and CD8+ populations showed that most T cells had the phenotypeof naive CD45RA+ T cells (Figure 3A). We also assessed whetherT cells were being synthesized by measuring the level of T-cellantigen-receptor episomes. Intrathymic rearrangements of genesencoding T-cell antigen receptors cause the formation of extrachromosomalDNA episomes, which mark T cells that have recently emigratedfrom the thymus to the periphery. As shown in Figure 3B, T-cellantigen-receptor episomes in Patients 1, 2, and 4 were firstdetected between day 60 and day 90, reached values found inage-matched controls, and remained stable for up to two yearsafter gene transfer. Thirteen months after treatment, Patient5 had 5500 CD45RA+ CD4+ T cells per cubic millimeter and 21,000T-cell antigen-receptor episomes per 100,000 peripheral-bloodmononuclear cells, respectively. These data correlated wellwith the development of a normal-sized thymus, as evaluatedby ultrasonography (in Patients 1, 2, 4, and 5) and by magneticresonance imaging in Patient 5 (respective size at one yearor more, 23 by 15 by 11.5 mm, 21 by 13 by 10 mm, 27 by 34 by13 mm, and 19 by 15 by 7 mm) (Figure 3C).
Figure 3. Numbers of Naive (CD45RA+) and Memory (CD45RO+) T Cells (Panel A) and Numbers of T-Cell Antigen-Receptor Episomes (Panel B) after Gene Therapy in Patients 1, 2, and 4 and Magnetic Resonance Image of a Coronal Section of the Thymus in Patient 5 Five Months after Gene Therapy (Panel C).
In Panel A, phenotypic quantification of naive and memory CD4+ T cells was performed with the use of double staining with fluorochrome-conjugated antibodies against CD4 and CD45RA or CD45RO. In Panel B, numbers of T-cell antigen-receptor episomes in peripheral-blood mononuclear cells were evaluated at different times. The normal range of T-cell antigen-receptor episomes for age-matched controls is 2500 to 20,000 per 100,000 peripheral-blood mononuclear cells. Arrows in Panel C show a normal-sized thymus after reconstitution of T cells.
Expression of 17 V families of T-cell receptors in Patients1, 2, 4, and 5 was similar to that in age-matched controls,and in these patients CD4+ and CD8+ T-cell populations remainedstable. In all patients, a gaussian distribution of the lengthsof complementarity-determining region 3 for 22 tested V familiesof T-cell receptors was observed (see Supplementary Appendix 1).
Capacity for T-Cell Proliferation
At the last follow-up visit, T cells from Patients 1, 2, 4,and 5 exhibited normal proliferative responses to in vitro stimulationwith phytohemagglutinin and anti-CD3 antibody (see Supplementary Appendix 1).Antigen-specific proliferative T-cell responseswere also observed after immunization of those four patientswith tetanus toxoid and polioviruses (see Supplementary Appendix 1).The addition of interleukin-2 to T cells from Patients 4and 5 enhanced in vitro proliferative responses to tetanus toxoid.T cells from Patient 1, who was immunized with bacille Calmette–Guérinat two months of age, also had a proliferative response to tuberculin(purified protein derivative).
Development of Natural Killer Cells
Natural killer cells became detectable 15 to 45 days after genetherapy in Patients 2, 4, and 5 and 150 days after gene therapyin Patient 1 (Figure 4). In Patients 2 and 4, and to a lessermagnitude in Patient 5, the levels of natural killer cells peakedtwo to four months after gene therapy and then gradually decreased.In Patient 3, natural killer cells were also detected in theblood beginning on day 45. These cells expressed c as detectedby immunofluorescence analysis (see Supplementary Appendix 1)and exhibited cytotoxic activity against K562 target cells (datanot shown).
Figure 4. Absolute Numbers of CD56+ and CD16+ Cells per Cubic Millimeter of Whole Blood after Gene Therapy in Patients 1 through 5.
Serum Immunoglobulins and Antibody Production
Serum IgG, IgA, and IgM levels at 25, 21, and 13 months in Patients1, 2, and 5, respectively, were within the age-related normalrange (Figure 5). Low IgG and IgA levels persisted in Patient4 (Figure 5). Antibodies against tetanus toxoid, diphtheriatoxoid, and poliovirus antigens were first found one month afterthe third immunization (Table 2) and persisted for more thansix months in Patients 1, 2, and 4. Antibodies against S. pneumoniaein Patient 2 and H. influenzae in Patient 1 and Patient 2 werealso detected. In contrast, immunization of Patient 5 failedto elicit an antibody response. Isohemagglutinins were consistentlydetected in the serum of Patients 1, 2, and 4 one year or moreafter gene therapy (Table 2). In three patients, the percentageof CD27+ and CD19+ B cells was similar to that of age-matchedcontrols (see Supplementary Appendix 1).
Table 2. Peak Antibody Responses after Immunization.
Integration and Expression of c Provirus
In Patients 1, 2, 4, and 5, all CD3+ T cells carried the c transgene,as compared with 1 to 5 percent of B cells, 0.05 to 2 percentof monocytes, and 0.05 to 0.5 percent of granulocytes (Figure 2).The frequency of c-containing T cells, B cells, monocytes,and granulocytes was stable during the study period (Figure 2).In Patients 2, 4, and 5, the presence of the c gene coincidedwith the expression of c chains (see Supplementary Appendix 1).In bone marrow samples obtained from Patient 2 and Patient4 21 and 13 months, respectively, after gene transfer, 1 to5 percent of colony-forming units derived from cultured CD34+cells contained the transgene (frequency of long-term-cultureinitiating cells, 1:1000 in Patient 2 and 1:500 in Patient 4)(data not shown).
Patient 3
Reconstitution of T cells failed to occur in Patient 3 (Figure 1),despite the presence of c-positive cells, as detected byPCR and immunofluorescence analysis of peripheral-blood mononuclearcells from day 30 up to four months after gene transfer. Aftersplenectomy, a strong c signal was detected among sorted CD19+and CD16+ cells by nonquantitative PCR analysis. There wereno CD3+ T cells in the spleen, and provirus (i.e., vector) wasnot detected in a bone marrow sample obtained at the time ofsplenectomy.
Discussion
We found that four of five patients with X-linked severe combinedimmunodeficiency due to a deficiency of the c chain who weretreated with autologous CD34+ cells from bone marrow that hadbeen transduced ex vivo with the c gene showed evidence of afunctional immune system and sustained clinical benefit. Theseresults extend a preliminary report of two patients treatedin this way.7 The gene-therapy protocol we used is safe, andno evidence of the emergence of a replication-competent retrovirushas been detected.
The evidence that virtually all T cells and natural killer cellsbut fewer B cells and myeloid cells were transduced suggeststhat c expression gives progenitors of T cells and natural killercells a selective growth advantage. Since transduced monocytes,granulocytes, and colonies derived from long-term cultures oftransduced CD34+ cells were consistently detected one to twoyears after gene transfer, it is likely that long-lived immatureprogenitor cells were targeted by the vector. Moreover, thepersistence of T-cell antigen-receptor episomes,11,12 naiveT cells, and the development of a normal-sized thymus indicateongoing formation of T cells and thymopoiesis, which most likelyoriginated from transduced CD34+ progenitors. These findingssuggest that both committed myeloid and lymphoid progenitorcells were transduced (implying that these cells persist inthe bone marrow for at least one to two years) or that uncommittedpluripotent progenitor cells were transduced by the c-containingvector. Evaluation of provirus integration sites in myeloidand lymphoid cells14,15 should help clarify this issue.
In our four successfully treated patients, the pattern of restorationof T cells differed from that observed after transplantationof haploidentical hematopoietic stem cells in patients withsevere combined immunodeficiency.3,4 After the latter, T cellsusually begin to appear within four to six months, and the numberof T cells in peripheral blood rarely exceeds 2000 per cubicmillimeter.3,4 In contrast, after gene therapy, T cells appearedwithin two to four months, at levels of 2000 to 8000 per cubicmillimeter. The absence of graft-versus-host disease and theex vivo activation of CD34+ cells with cytokines could havecontributed to the rapid reconstitution.
The characteristics of the T cells in the four patients weresimilar to those of age-matched controls. The diversity of T-cellreceptors and the presence of T-cell antigen-receptor episomesand naive T cells suggest that the T cells after gene therapyderived from genuine thymopoiesis and not from an increase inthe number of transduced mature T cells.16,17 It is interestingthat neither membrane expression of a truncated c protein (inPatient 1) nor the presence of numerous maternal T cells (inPatient 5) influenced the development of T cells. Although cgene transfer did not increase B-cell numbers substantially,enough immunoglobulin was produced to avoid the need for intravenousimmune globulin. It is not known whether the few transducedB cells account for the production of antibodies in these patientsor whether nontransduced B cells are also involved.18 Sincethere were more detectable memory B cells (CD27+ and CD19+)than transduced B cells, it is possible that c-negative B cellsretain some function.
In conclusion, our study demonstrates that the infusion of autologousc-transduced cells, despite the low efficiency of the transductionprocess, can repair the immune system in patients with X-linkedsevere combined immunodeficiency. Although the repair is incomplete,it is sufficient to provide protective immunity. Despite anobvious requirement for long-term assessment and further analysisin a larger cohort of patients, these results suggest that asimilar approach could be used for other forms of severe combinedimmunodeficiency.19,20,21,22,23,24
Supported by grants from INSERM, Association Françaisecontre les Myopathies, Programme Hospitalier de Recherche Cliniqueof the Health Ministry (AOM 0093), Assistance Publique–Hôpitauxde Paris, the Jeffrey Modell Foundation, and Fondation LouisJeantet (Geneva).
We are indebted to the families of the patients for their continuoussupport of the study; to the medical and nursing staff of theUnité d'Immunologie et d'Hématologie Pédiatriques,Hôpital des Enfants Malades, for patient care; to Jean-LaurentCasanova, Geneviève de Saint Basile, and Anne Durandyfor their contribution to the study; to L. Coulombel for helpfuladvice; to F. Gross, P. Nussbaum, C. Harre, C. Jacques, andF. Selz for technical help; to S. Yoshimura and I. Kato (TakaraShugo, Shiga, Japan) for providing the CD-296 fibronectin fragment;to B. Bussière, C. Caillot, and J. Caraux (Amgen, France)for providing stem-cell factor and megakaryocyte growth anddevelopment factor; and to P. Johnson and D. Louis for editorialassistance.
Source Information
From the Laboratoire INSERM (S.H.-B.-A., F.D., C.H., J.-P.V., A.F., M.C.-C.), the Laboratoire de Thérapie Cellulaire et Génique (S.H.-B.-A., F.C., C.H., M.C.-C.), the Laboratoire d'Immunologie Pédiatrique (F.D.), and Unité d'Immunologie et d'Hématologie Pédiatriques (S.D.-G., A.F.), Hôpital Necker Enfants Malades, Paris; Unité de Biologie du Gène, Institut Pasteur, Paris (C.B.); the Molecular Immunology Unit, Institute of Child Health, London (A.J.T.); the Department of Immunology and Hematology, Wilhelmina Kinderziekenhuis Lundlaan, Utrecht, the Netherlands (N.W.); and the Department of Pediatrics, Louisiana State University Health Science Center, New Orleans (R.S.). Other authors were E. Graham Davies, M.D., Great Ormond Street Hospital for Children, National Health Service Trust, London; Wietse Kuis, M.D., Ph.D., Department of Immunology and Hematology, Wilhelmina Kinderziekenhuis Lundlaan, Utrecht, the Netherlands; and Lilly Leiva, Ph.D., Department of Pediatrics, Louisiana State University Health Science Center, New Orleans.
Address reprint requests to Dr. Cavazzana-Calvo at the Laboratoire de Thérapie Cellulaire et Génique, Hôpital Necker Enfants Malades, 149 rue de Sèvres, 75015 Paris, France, or at cavazzan{at}necker.fr.
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(2008). Sustained Expression of {alpha}1-Antitrypsin after Transplantation of Manipulated Hematopoietic Stem Cells. Am. J. Respir. Cell Mol. Bio.
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(2008). Tolerization of a Type I Allergic Immune Response through Transplantation of Genetically Modified Hematopoietic Stem Cells. J. Immunol.
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(2008). Development of gene therapy for blood disorders. Blood
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(2008). Retroviral Vectors for Gene Transfer. CSH Protocols
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(2008). An experimental system for the evaluation of retroviral vector design to diminish the risk for proto-oncogene activation. Blood
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Deschamps, M., Mercier-Lethondal, P., Certoux, J. M., Henry, C., Lioure, B., Pagneux, C., Cahn, J. Y., Deconinck, E., Robinet, E., Tiberghien, P., Ferrand, C.
(2007). Deletions within the HSV-tk transgene in long-lasting circulating gene-modified T cells infused with a hematopoietic graft. Blood
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(2007). Site-directed transposon integration in human cells. Nucleic Acids Res
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Dunbar, C. E.
(2007). The Yin and Yang of Stem Cell Gene Therapy: Insights into Hematopoiesis, Leukemogenesis, and Gene Therapy Safety. ASH Education Book
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(2006). Complete correction of murine Artemis immunodeficiency by lentiviral vector-mediated gene transfer. Proc. Natl. Acad. Sci. USA
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Shou, Y., Ma, Z., Lu, T., Sorrentino, B. P.
(2006). Unique risk factors for insertional mutagenesis in a mouse model of XSCID gene therapy. Proc. Natl. Acad. Sci. USA
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(2006). Gene therapy and uterine leiomyoma: a review. Hum Reprod Update
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Lore, K., Seggewiss, R., Guenaga, F. J., Pittaluga, S., Donahue, R. E., Krouse, A., Metzger, M. E., Koup, R. A., Reilly, C., Douek, D. C., Dunbar, C. E.
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(2006). Genome-wide mapping of foamy virus vector integrations into a human cell line.. J. Gen. Virol.
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(2006). Correction of canine X-linked severe combined immunodeficiency by in vivo retroviral gene therapy. Blood
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Nagasawa, Y., Wood, B. L., Wang, L., Lintmaer, I., Guo, W., Papayannopoulou, T., Harkey, M. A., Nourigat, C., Blau, C. A.
(2006). Anatomical Compartments Modify the Response of Human Hematopoietic Cells to a Mitogenic Signal. Stem Cells
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(2006). Gene Transfer to Corneal Epithelium and Keratocytes Mediated by Ultrasound with Microbubbles. IOVS
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Kirschner, L. S.
(2006). Emerging Treatment Strategies for Adrenocortical Carcinoma: A New Hope. J. Clin. Endocrinol. Metab.
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(2006). Long-term immune reconstitution in RAG-1-deficient mice treated by retroviral gene therapy: a balance between efficiency and toxicity. Blood
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Lippin, Y., Dranitzki-Elhalel, M., Brill-Almon, E., Mei-Zahav, C., Mizrachi, S., Liberman, Y., Iaina, A., Kaplan, E., Podjarny, E., Zeira, E., Harati, M., Casadevall, N., Shani, N., Galun, E.
(2005). Human erythropoietin gene therapy for patients with chronic renal failure. Blood
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Santat, L., Paz, H., Wong, C., Li, L., Macer, J., Forman, S., Wong, K. K., Chatterjee, S.
(2005). Recombinant AAV2 transduction of primitive human hematopoietic stem cells capable of serial engraftment in immune-deficient mice. Proc. Natl. Acad. Sci. USA
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Lucas, M. L., Seidel, N. E., Porada, C. D., Quigley, J. G., Anderson, S. M., Malech, H. L., Abkowitz, J. L., Zanjani, E. D., Bodine, D. M.
(2005). Improved transduction of human sheep repopulating cells by retrovirus vectors pseudotyped with feline leukemia virus type C or RD114 envelopes. Blood
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Seggewiss, R., Dunbar, C. E.
(2005). Old before its time: age-related thymic dysfunction may preclude efficacy of gene therapy in older SCID-X1 patients. Blood
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Thrasher, A. J., Hacein-Bey-Abina, S., Gaspar, H. B., Blanche, S., Davies, E. G., Parsley, K., Gilmour, K., King, D., Howe, S., Sinclair, J., Hue, C., Carlier, F., von Kalle, C., de Saint Basile, G., le Deist, F., Fischer, A., Cavazzana-Calvo, M.
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(2004). Diminished Mobilization of Self-Inactivating (SIN) Lentiviral Vectors Containing Globin Regulatory Elements Compared to Those Containing a Retroviral Long Terminal Repeat.. ASH ANNUAL MEETING ABSTRACTS
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(2004). Autoimmune Lymphoproliferative Syndrome with Somatic Fas Mutations. NEJM
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Asheuer, M., Pflumio, F., Benhamida, S., Dubart-Kupperschmitt, A., Fouquet, F., Imai, Y., Aubourg, P., Cartier, N.
(2004). Human CD34+ cells differentiate into microglia and express recombinant therapeutic protein. Proc. Natl. Acad. Sci. USA
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(2004). Transient in vivo selection of transduced peripheral blood cells using antifolate drug selection in rhesus macaques that received transplants with hematopoietic stem cells expressing dihydrofolate reductase vectors. Blood
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Brown, V. I., Fang, J., Alcorn, K., Barr, R., Kim, J. M., Wasserman, R., Grupp, S. A.
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Geronimi, F., Richard, E., Redonnet-Vernhet, I., Lamrissi-Garcia, I., Lalanne, M., Ged, C., Moreau-Gaudry, F., de Verneuil, H.
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Alexander, D R
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Cooper, R. S., Psaty, B. M.
(2003). Genomics and Medicine: Distraction, Incremental Progress, or the Dawn of a New Age?. ANN INTERN MED
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(2003). Retroviral transduction efficiency of G-CSF+SCF-mobilized peripheral blood CD34+ cells is superior to G-CSF or G-CSF+Flt3-L-mobilized cells in nonhuman primates. Blood
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Sarzotti, M., Patel, D. D., Li, X., Ozaki, D. A., Cao, S., Langdon, S., Parrott, R. E., Coyne, K., Buckley, R. H.
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Hemminki, A., Kanerva, A., Liu, B., Wang, M., Alvarez, R. D., Siegal, G. P., Curiel, D. T.
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Cooper, M. D., Lanier, L. L., Conley, M. E., Puck, J. M.
(2003). Immunodeficiency Disorders. ASH Education Book
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Yates, F., Malassis-Seris, M., Stockholm, D., Bouneaud, C., Larousserie, F., Noguiez-Hellin, P., Danos, O., Kohn, D. B., Fischer, A., de Villartay, J.-P., Cavazzana-Calvo, M.
(2002). Gene therapy of RAG-2-/- mice: sustained correction of the immunodeficiency. Blood
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Neff, T., Horn, P. A., Valli, V. E., Gown, A. M., Wardwell, S., Wood, B. L., von Kalle, C., Schmidt, M., Peterson, L. J., Morris, J. C., Richard, R. E., Clackson, T., Kiem, H.-P., Blau, C. A.
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Handgretinger, R., Koscielniak, E., Niethammer, D., Cavazzana-Calvo, M., Hacein-Bey-Abina, S., Fischer, A.
(2002). Gene Therapy for Severe Combined Immunodeficiency Disease. NEJM
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Hawley, R. G., Sobieski, D. A.
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Rosen, F. S.
(2002). Successful Gene Therapy for Severe Combined Immunodeficiency. NEJM
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Walters, M. C., Nienhuis, A. W., Vichinsky, E.
(2002). Novel Therapeutic Approaches in Sickle Cell Disease. ASH Education Book
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A correction has been published: N Engl J Med 2002;347(8):613.
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or 
receptors, and the numbers of CD4+ and CD8+ T cells were similar to those in age-matched controls (data not shown). Conversely, no T cells were detected in the blood of Patient 3 up to six months after treatment (Figure 1). 
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