Contribution of the use of basic telemedicine tools to the care of children and adolescents with juvenile idiopathic arthritis at the Puerto Montt Hospital, Chile. / Aporte del uso de herramientas básicas de Telemedicina en la atención de niños y adolescentes con Artritis idiopática juvenil, en el Hospital de Puerto Montt. Chile.

Children and adolescents with rheumatologic diseases require specialized and comprehensive care, but pediatric rheumatologists and immunologists are concentrated in hospitals with specific, high-cost and modern technology. Considering that some patients with juvenile idiopathic arthritis (JIA) live in rural, remote and limited accessibility areas, the use of Telemedicine (TM) can optimize diag nosis, follow-up and prognosis. OBJECTIVE: Reporting 10 years of experience of a mixed care model: face-to-face and distance, using basic TM; the institutional impact, advantages, disadvantages and acceptance informed by parents and patients. PATIENTS AND METHOD: Exploratory, descriptive, and re trospective study with qualitative component. After the authorization of a scientific-ethics committee of the Reloncaví Health Service and the application of informed consent, a review of medical records was carried out and a qualitative survey was applied to parents and children over 14 years of age with JIA, seen between 2005-2015 in the pediatric ambulatory rheumatology polyclinic of Puerto Montt Hospital. RESULTS: The were 27/35 participating patients with JIA attended by a trained pediatrician and assisted by distance (1,000 km) by an immunologist. The 8/35 patients did not answer by choice or change of address. The 70% of parents and patients accepted the model of care and 4% would pre fer sporadic care only by specialists for diagnosis and follow-up. The number of patients transferred annually decreased from 10 to 1. The advantages of the care model outweighed the disadvantages perceived by parents and JIA patients. CONCLUSION: The use of TM tools in JIA decreased transfers, improved follow-up and were considered advantageous by patients and their parents.

Aporte del uso de herramientas básicas de Telemedicina en la atención de niños y adolescentes con Artritis idiopática juvenil, en el Hospital de Puerto Montt. Chile / Contribution of the use of basic telemedicine tools to the care of children and adolescents with juvenile idiopathic arthritis at the Puerto Montt Hospital, Chile

Resumen: Niños y adolescentes con enfermedades reumatológicas, requieren atención especializada e integral, sin embargo, reumatólogos e inmunólogos pediátricos se concentran en hospitales con tecnología específica, costosa y moderna. Como algunos pacientes con Artritis idiopática juvenil (AIJ) vive en áreas rurales, lejanas y de accesibilidad limitada, el uso de Telemedicina (TM) puede optimizar el diagnóstico, seguimiento y pronóstico. Objetivo: Mostrar 10 años de experiencia de un modelo de atención mixta: presencial y a distancia, usando TM básica; el impacto institucional, ventajas, des ventajas y aceptación reportados por padres y pacientes. Pacientes y Método: Estudio exploratorio, descriptivo, retrospectivo con componente cualitativo. Previa autorización de comité ético-científico del Servicio de salud del Reloncaví y la aplicación de consentimiento/asentimiento informado, se efectuó revisión de historias clínicas y se aplicó encuesta cualitativa a padres y niños mayores de 14 años con AIJ, atendidos entre 2005-2015 en el policlínico de reumatología infantil Hospital Puerto Montt. Resultados: Participaron 27/35 pacientes con AIJ atendidos por pediatra capacitado, aseso rado a distancia (1.000 km) por inmunólogo. 8/35 pacientes no contestaron por opción o cambio de domicilio. 70 % de padres y pacientes aceptaron el modelo de atención y 4% preferirían atención esporádica solo por especialista para diagnóstico y seguimiento. El número de pacientes trasladados anualmente disminuyó de 10 a 1. Las ventajas del modelo de atención superaron las desventajas per cibidas por padres y pacientes con AIJ. Conclusión: El uso de herramientas de TM en AIJ disminuyó los traslados, mejoró el seguimiento y fue considerado ventajoso por los padres y pacientes.

Abstract: Children and adolescents with rheumatologic diseases require specialized and comprehensive care, but pediatric rheumatologists and immunologists are concentrated in hospitals with specific, high-cost and modern technology. Considering that some patients with juvenile idiopathic arthritis (JIA) live in rural, remote and limited accessibility areas, the use of Telemedicine (TM) can optimize diag nosis, follow-up and prognosis. Objective: Reporting 10 years of experience of a mixed care model: face-to-face and distance, using basic TM; the institutional impact, advantages, disadvantages and acceptance informed by parents and patients. Patients and Method: Exploratory, descriptive, and re trospective study with qualitative component. After the authorization of a scientific-ethics committee of the Reloncaví Health Service and the application of informed consent, a review of medical records was carried out and a qualitative survey was applied to parents and children over 14 years of age with JIA, seen between 2005-2015 in the pediatric ambulatory rheumatology polyclinic of Puerto Montt Hospital. Results: The were 27/35 participating patients with JIA attended by a trained pediatrician and assisted by distance (1,000 km) by an immunologist. The 8/35 patients did not answer by choice or change of address. The 70% of parents and patients accepted the model of care and 4% would pre fer sporadic care only by specialists for diagnosis and follow-up. The number of patients transferred annually decreased from 10 to 1. The advantages of the care model outweighed the disadvantages perceived by parents and JIA patients. Conclusion: The use of TM tools in JIA decreased transfers, improved follow-up and were considered advantageous by patients and their parents.

Antagonism between granulocytic maturation and deacetylase inhibitor-induced apoptosis in acute promyelocytic leukaemia cells.

BACKGROUND: Transcriptional repression is a key mechanism driving leukaemogenesis. In acute promyelocytic leukaemia (APL), the fusion protein promyelocytic leukaemia-retinoic acid receptor-α fusion (PML-RARα) recruits transcriptional repressors to myeloid differentiation genes. All-trans-retinoic acid (ATRA) induces the proteasomal degradation of PML-RARα and granulocytic differentiation. Histone deacetylases (HDACs) fall into four classes (I-IV) and contribute to the transcription block caused by PML-RARα. METHODS: Immunoblot, flow cytometry, and May-Grünwald-Giemsa staining were used to analyze differentiation and induction of apoptosis. RESULTS: A PML-RARα- and ATRA-dependent differentiation programme induces granulocytic maturation associated with an accumulation of the myeloid transcription factor CCAAT/enhancer binding protein (C/EBP)ɛ and of the surface protein CD11b. While this process protects APL cells from inhibitors of class I HDAC activity, inhibition of all Zinc-dependent HDACs (classes I, II, and IV) with the pan-HDACi (histone deacetylase inhibitor(s)) LBH589 induces apoptosis of immature and differentiated APL cells. LBH589 can eliminate C/EBPɛ and the mitochondrial apoptosis regulator B-cell lymphoma (BCL)-xL in immature and differentiated NB4 cells. Thus, BCL-xL and C/EBPɛ are newly identified molecular markers for the efficacy of HDACi against APL cells. CONCLUSIONS: Our results could explain the therapeutic limitations occurring with ATRA and class I HDACi combinations. Pro-apoptotic effects caused by pan-HDAC inhibition are not blunted by ATRA-induced differentiation and may provide a clinically interesting alternative.

Activating c-KIT mutations confer oncogenic cooperativity and rescue RUNX1/ETO-induced DNA damage and apoptosis in human primary CD34+ hematopoietic progenitors.

The RUNX1/ETO (RE) fusion protein, which originates from the t(8;21) chromosomal rearrangement, is one of the most frequent translocation products found in de novo acute myeloid leukemia (AML). In RE leukemias, activated forms of the c-KIT tyrosine kinase receptor are frequently found, thereby suggesting oncogenic cooperativity between these oncoproteins in the development and maintenance of t(8;21) malignancies. In this report, we show that activated c-KIT cooperates with a C-terminal truncated variant of RE, REtr, to expand human CD34+ hematopoietic progenitors ex vivo. CD34+ cells expressing both oncogenes resemble the AML-M2 myeloblastic cell phenotype, in contrast to REtr-expressing cells which largely undergo granulocytic differentiation. Oncogenic c-KIT amplifies REtr-depended clonogenic growth and protects cells from exhaustion. Activated c-KIT reverts REtr-induced DNA damage and apoptosis. In the presence of activated c-KIT, REtr-downregulated DNA-repair genes are re-expressed leading to an enhancement of DNA-repair efficiency via homologous recombination. Together, our results provide new mechanistic insight into REtr and c-KIT oncogenic cooperativity and suggest that augmented DNA repair accounts for the increased chemoresistance observed in t(8;21)-positive AML patients with activated c-KIT mutations. This cell-protective mechanism might represent a new therapeutic target, as REtr cells with activated c-KIT are highly sensitive to pharmacological inhibitors of DNA repair.

Histone arginine methylation keeps RUNX1 target genes in an intermediate state.

The coordinated recruitment of epigenetic regulators of gene expression by transcription factors such as RUNX1 (AML1, acute myeloid leukemia 1) is crucial for hematopoietic differentiation. Here, we identify protein arginine methyltransferase 6 (PRMT6) as a central functional component of a RUNX1 corepressor complex containing Sin3a and HDAC1 in human hematopoietic progenitor cells. PRMT6 is recruited by RUNX1 and mediates asymmetric histone H3 arginine-2 dimethylation (H3R2me2a) at megakaryocytic genes in progenitor cells. H3R2me2a keeps RUNX1 target genes in an intermediate state with concomitant H3K27me3 and H3K4me2 but not H3K4me3. Upon megakaryocytic differentiation PRMT6 binding is lost, the H3R2me2a mark decreases and a coactivator complex containing WDR5/MLL and p300/pCAF is recruited. This leads to an increase of H3K4me3 and H3K9ac, which result in augmented gene expression. Our results provide novel mechanistic insight into how RUNX1 activity in hematopoietic progenitor cells maintains differentiation genes in a suppressed state but poised for rapid transcriptional activation.

Overexpression of the anti-apoptotic protein AVEN contributes to increased malignancy in hematopoietic neoplasms.

AVEN has been identified as an inhibitor of apoptosis, which binds to the adaptor protein, APAF-1, and thereby prevents apoptosome formation and mitochondrial apoptosis. Recent data have demonstrated high expression levels of AVEN messenger RNA in acute leukemias as well as a positive correlation between AVEN mRNA overexpression and poor prognosis in childhood acute lymphoblastic leukemia. On the basis of these data, we investigated the potential involvement of AVEN in tumorigenesis. First, we confirmed the overexpression of AVEN in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) patient samples. We then established a transgenic mouse model with T-cell-specific overexpression of AVEN, with which we demonstrated the oncogenic cooperation of AVEN with heterozygous loss of p53. Finally, we used a subcutaneous xenograft mouse model to show that AVEN knockdown in the T-ALL cell lines, MOLT-4 and CCRF-CEM, and in the acute myeloblastic leukemia cell line, Kasumi-1, leads to a halt in tumor growth owing to the increased apoptosis and decreased proliferation of tumor cells. Collectively, our data demonstrate that the anti-apoptotic molecule, AVEN, functions as an oncoprotein in hematopoietic neoplasms.

Physiological regulation of transgene expression by a lentiviral vector containing the A2UCOE linked to a myeloid promoter.

Protection against epigenetic silencing is a desirable feature of future gene therapy vectors, in particular for those applications in which transgene expression will not confer growth advantage to gene-transduced cells. The ubiquitous chromatin opening element (UCOE) consisting of the methylation-free CpG island encompassing the dual divergently transcribed promoters of the human HNRPA2B1-CBX3 housekeeping genes (A2UCOE) has been shown to shield constitutive active heterologous promoters from epigenetic modifications and chromosomal position effects. However, it is unclear if this element can be used to improve expression from tissue-specific enhancer/promoters, while maintaining tissue specificity in hematopoietic cells. Here, we evaluated the potential of the A2UCOE in combination with the myeloid-specific myeloid related protein 8 (MRP8) promoter to target transgene expression specifically to myeloid cells in vitro and in vivo from a self-inactivating lentiviral vector. The inclusion of the A2UCOE did not interfere with specific upregulation of MRP8 promoter activity during myeloid differentiation and mediated sustained and vector copy-dependent expression in myeloid cells. Notably, the A2UCOE did not protect the MRP8 promoter from methylation in the P19 embryonal carcinoma cell line, suggesting that this element maintains the inherent epigenetic state and transcriptional activity of cellular promoters in their native configuration. Thus, the A2UCOE could represent a useful protective genetic element in gene therapy vectors, ensuring physiological transcriptional regulation of tissue-specific promoters independent of the chromosomal integration site.

A new PG13-based packaging cell line for stable production of clinical-grade self-inactivating gamma-retroviral vectors using targeted integration.

The clinical application of self-inactivating (SIN) retroviral vectors has been hampered by the lack of reliable and efficient vector production technologies. To enable production of SIN gamma-retroviral vectors from stable producer clones, a new PG13-based packaging cell, known as PG368, was developed. Viral vector expression constructs can be reliably inserted at a predefined genomic locus of PG368 packaging cells by an Flp-recombinase-mediated targeted cassette exchange (RMCE) reaction. A new, carefully designed vector-targeting construct, pEMTAR-1, eliminated the co-packaging of the selectable marker gene used for the identification of successful recombination at the predefined genomic locus and thus, improved the safety of the production system. Selected clones produced vector supernatants at consistent titers. The targeted insertion of therapeutically relevant SIN vectors for chronic granulomatous disease and X-linked severe combined immunodeficiency into PG368 cells results in stable titers within the range necessary for clinical application. The production of retroviral SIN vectors from stable clinical-grade producer cells is feasible and will contribute to the safe production and application of SIN gamma-retroviral vectors for clinical trials.

Retroviral vector integration in post-transplant hematopoiesis in mice conditioned with either submyeloablative or ablative irradiation.

X-linked chronic granulomatous disease (X-CGD) is an inherited immunodeficiency with absent phagocyte NADPH-oxidase activity caused by defects in the gene-encoding gp91(phox). Here, we evaluated strategies for less intensive conditioning for gene therapy of genetic blood disorders without selective advantage for gene correction, such as might be used in a human X-CGD protocol. We compared submyeloablative with ablative irradiation as conditioning in murine X-CGD, examining engraftment, oxidase activity and vector integration in mice transplanted with marrow transduced with a gamma-retroviral vector for gp91(phox) expression. The frequency of oxidase-positive neutrophils in the donor population was unexpectedly higher in many 300 cGy-conditioned mice compared with lethally irradiated recipients, as was the fraction of vector-marked donor secondary CFU-S12. Vector integration sites in marrow, spleen and secondary CFU-S12 DNA from primary recipients were enriched for cancer-associated genes, including Evi1, and integrations in or near cancer-associated genes were more frequent in marrow and secondary CFU-S12 from 300 cGy-conditioned mice compared with fully ablated mice. These findings support the concept that vector integration can confer a selection bias, and suggest that the intensity of the conditioning regimen may further influence the effects of vector integration on clonal selection in post-transplant engraftment and hematopoiesis.

Transgene optimization significantly improves SIN vector titers, gp91phox expression and reconstitution of superoxide production in X-CGD cells.

Gene therapy has proven to be of potential value for the correction of inherited hematopoietic disorders. However, the occurrence of severe side effects in some of the clinical trials has questioned the safety of this approach and has hampered the use of long terminal repeat-driven vectors for the treatment of a large number of patients. The development of self-inactivating (SIN) vectors with reduced genotoxicity provides an alternative to the currently used vectors. Our initial attempts to use SIN vectors for the correction of a myeloid disorder, chronic granulomatous disease, failed due to low vector titers and poor transgene expression. The optimization of the transgene cDNA (gp91(phox)) resulted in substantially increased titers and transgene expression. Most notably, transgene optimization significantly improved expression of a second cistron located downstream of gp91(phox). Thus, optimization of the transgene sequence results in higher expression levels and increased therapeutic index allowing the use of low vector copy numbers per transduced cell and weaker internal promoters.

T cells for suicide gene therapy: activation, functionality and clinical relevance.

In order to control graft-versus-host disease after donor lymphocyte infusion, T cells can be retrovirally transduced with a suicide gene. However, the immune competence of activated T cells appears compromised, responsible for reduced alloreactivity. The present study compared different activation protocols using soluble or bead-coupled antibodies regarding T-cell subtype expansion capacity and functionality. T cells were purified on a laboratory and clinical scale using both CD3 and CD4/CD8 antibodies for selection, leading to a mean purity of 96%. Transductions were performed with a GMP-grade CD34/HSV-TK vector. Activation with soluble CD3/CD28-antibodies +1000 U/ml IL-2 induced a 50-fold expansion of T cells over 14 days, whereas T cells activated with bead-coupled antibodies only expanded 2-4-fold restricted to the first week. Apart from using soluble antibodies, proliferation was highly IL-2 dependent. Expansion of CMV-specific T cells coincided with the expansion of whole CD3(+) cells. Soluble antibodies and higher IL-2 concentrations preferentially stimulated CD8(+) T cells, while bead-coupled antibodies +20 U/ml IL-2 preserved the CD4/CD8 ratio. Irrespective of the activation protocol, there was a shift from a naive to memory phenotype. When activated with soluble antibodies, mainly CD8(+) T cells were transduced. Furthermore, Th1/Th2 cytokine secretion was reduced. In contrast, CD4(+)/CD8(+) T cells activated with bead-coupled antibodies were rather homogenously transduced and cytokine secretion did not appear to be affected.

Progress and prospects: gene therapy clinical trials (part 1).

Over the last two decades gene therapy has moved from preclinical to clinical studies for many diseases ranging from single gene disorders such as cystic fibrosis and Duchenne muscular dystrophy, to more complex diseases such as cancer and cardiovascular disorders. Gene therapy for severe combined immunodeficiency (SCID) is the most significant success story to date, but progress in many other areas has been significant. We asked 20 leaders in the field succinctly to summarize and comment on clinical gene therapy research in their respective areas of expertise and these are published in two parts in the Progress and Prospect series.

Kinetics of cell death in T lymphocytes genetically modified with two novel suicide fusion genes.

Donor lymphocyte infusions (DLI) following allogeneic stem cell transplantation are known to mediate graft-versus-leukemia effect (GVL). A major side effect of these immunotherapies is the development of graft-versus-host diseases (GVHD). One promising approach to prevent GVHD is to genetically modify donor T cells with a suicide mechanism that can be induced in the case of GVHD. Here we report on a retroviral vector containing the death effector domain (DED) of the human Fas-associated protein with death domain (FADD). The DED was fused to two copies of an FKBP506-binding protein and a truncated version of the human low-affinity receptor for nerve growth factor (LNGFR). Activation of the death signal pathway can be triggered upon the addition of chemical inducers of dimerization. This construct was functionally compared to an optimized HSV-TK vector in which a hypersensitive mutant of the herpes simplex virus thymidine kinase gene (TK39) was fused to a cytoplasmic truncated version of the cell surface antigen CD34. A direct comparison between both vectors in primary T lymphocytes showed that the number of T cells transduced with vectors containing the DED was significantly reduced within 24 h of drug administration whereas ganciclovir treatment of TK39-transduced T cells showed a delay in cell death of approximately 3-4 days. Our results indicate that constructs containing the DED may prove to be the most efficient mechanism to quickly eliminate alloreactive T cells.

Efficient gene transfer into the CNS by lentiviral vectors purified by anion exchange chromatography.

Lentiviral vectors have been shown to stably transduce dividing and non-dividing target cells in vitro and in vivo. However, in vivo gene transfer applications with viral vectors in the central nervous system require highly efficient vector preparations, because only very small volumes can be injected stereotactically without damage to the brain tissue. Since lentiviral vectors are generated in transient co-transfection systems, viral preparations need to be purified and efficiently concentrated before injection into the brain. We describe an alternative procedure to concentrate lentiviral preparations by binding viral particles to an anion exchange column. Viral particles are eluted with sodium chloride, desalted and further concentrated by ultrafiltration. These vector preparations allowed high levels of gene transfer into terminally differentiated neuronal and glial cells and long-term transgene expression without any signs of acute and long-term toxicity or inflammation. The purification of lentiviral vectors from large-scale preparations by anion exchange chromatography allowed us to concentrate the virus to small volumes and to use these preparations to genetically modified target cells in vivo without signs of acute inflammatory responses.

Generation and characterization of human hematopoietic cell lines expressing factor VIII.

Considering the plasticity of hematopoietic stem cells (HSC), they would be ideal targets for gene therapy of hemophilia A by virtue of their progeny providing immediate access to the bloodstream. However, several attempts to show expression of recombinant factor VIII (rFVIII) by primary hematopoietic cells and cell lines have failed; this failure was attributed to the inability of HSC to secrete rFVIII. Here we describe the generation of stable, FVIII-secreting hematopoietic cell lines representing different blood-cell types using a bicistronic lentiviral vector encoding for a B-domain-deleted FVIII (FVIII Delta B) and enhanced green fluorescence protein (EGFP). Transduced cell lines with erythroid and/or megakaryocytic background, (K562-F8 and TF-1-F8) secrete high levels of FVIII in the order of 76.4 and 41.6 ng FVIII:C/ml, whereas moderate and low levels are observed in B lymphoblastoid Raji-F8 cells and the T leukemia line Jurkat-F8 which secrete 6.73 and 1.83 ng FVIII:C/ml, respectively. The capacity to secrete rFVIII appeared to depend on factors related to the cell lineage rather than on the transduction efficacy. Stimulation of transduced cells with the protein kinase C (PKC)-activator phorbol myristate acetate (PMA) resulted in a marked augmentation of rFVIII secretion and enhanced green fluorescent protein (EGFP). Incubation with 0.1 and 1 ng/ml PMA resulted in up to 2.7-fold (K562-F8, Raji-F8) and 1.8-fold (293T-F8) increased rFVIII secretion. The established cell lines should be helpful in further elucidating mechanisms that are able to improve FVIII secretion in hematopoietic cells on a post-translational level and suggest reanalysis of hematopoietic cells as target for gene therapy of hemophilia.

Quantitative determination of lentiviral vector particle numbers by real-time PCR.

Here, we describe a quantitative, DNA-based, real-time PCR approach to determine the number of lentivirus particles that are present in vector preparations. In this approach, the minus strong-stop cDNA fragment that is present in viral capsids serves as template for PCR. Using this technology, we found that only 0.1%-1% of the virus particles that are present in vector preparations are infectious. The approach described here is rapid, reliable, and simple in concept and can be used to estimate both vector particles in supernatants and the number of infectious particles. Also, this approach can easily be adapted to a high-throughput system by using 96-well plates and a 2-h running time.

Multiple regions of ETO cooperate in transcriptional repression.

In acute myeloid leukemias (AMLs) with t(8;21), the transcription factor AML1 is juxtaposed to the zinc finger nuclear protein ETO (Eight-Twenty-One), resulting in transcriptional repression of AML1 target genes. ETO has been shown to interact with corepressors, such as N-CoR and mSin3A to form complexes containing histone deacetylases. To define regions of ETO required for maximal repressor activity, we analyzed amino-terminal deletions in a transcriptional repression assay. We found that ETO mutants lacking the first 236 amino acids were not affected in their repressor activity, whereas a further deletion of 85 amino acids drastically reduced repressor function and high molecular weight complex formation. This latter mutant can still homodimerize and bind to N-CoR but shows only weak binding to mSin3A. Furthermore, we could show that a "core repressor domain" comprising nervy homology region 2 and its amino- and carboxyl-terminal flanking sequences recruits mSin3A and induces transcriptional repression. These results suggest that mSin3A and N-CoR bind to ETO independently and that both binding sites cooperate to maximize ETO-mediated transcriptional repression. Thus, ETO has a modular structure, and the interaction between the individual elements is essential for the formation of a stable repressor complex and efficient transcriptional repression.

MDR1 gene expression in NOD/SCID repopulating cells after retroviral gene transfer under clinically relevant conditions.

We have adapted a recently published protocol for retroviral gene transfer into hematopoietic cells [A. J. Schilz et al. (1998) Blood 92: 3163-3171] with respect to clinical requirements such as large-volume vector stock generation, adequate cell source, high cell numbers, and serum-free conditions. We present data on transduction efficacy and expression of the multidrug resistance 1 (MDR1) gene in human CD34(+) cells from mobilized peripheral blood (PB) mediated by a gibbon ape leukemia virus (GALV)-pseudotyped retroviral vector. Using a 1-day cytokine-mediated prestimulation, consisting of human interleukin (IL)-3, IL-6, stem cell factor (SCF), Flt-3 ligand (FL), and thrombopoietin (TPO), followed by a 3-day transduction procedure, we were able to detect up to 51% CD34(+) cells expressing MDR1. Xenotransplantation of transduced cells into NOD/LtSz-scid/scid (NOD/SCID) mice resulted in a mean engraftment level of 23% (0.1 to 87%). As shown by quantitative PCR analysis, a mean of 12.7% (range 0.3 to 55%) of the engrafted human cells in the bone marrow of chimeric mice contained the MDR1 cDNA. Furthermore, enhanced expression of MDR1 above control levels was detected in up to 15% of the engrafted human cell population. Our data suggest that NOD/SCID repopulating cells derived from mobilized PB can be transduced efficiently with existing retroviral vector systems under clinically applicable conditions.

Gene therapy of chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder which results from absence or malfunction of the respiratory burst oxidase normally expressed in neutrophils and other phagocytic leukocytes. Two-thirds of the patients are males hemizygous for mutations in the X-linked gene coding for gp91-phox. As a therapeutic approach towards the X-linked form of CGD bicistronic retroviral vectors containing the gp91-phox gene and a selectable marker gene were constructed. The ability of these vectors to restore NADPH oxidase activity was tested in a human myeloid leukemic cell line that is defective in superoxide production, as well as in primary CD34+ cells obtained from X-CGD patients. Under optimal conditions 80% of the CD34+ cells derived from bone marrow of one X-CGD patient were transduced. The level of superoxide production, in phagocytes derived from transduced cells was 68.9% of normal levels. Considering that low levels of superoxide generating activity are sufficient for normal host defense, the present experiments provide the basis for the development of a gene replacement therapy for the X-linked form of CGD.