OVERVIEW: How Do We Translate Gene Therapy to Clinical Trials?

Transcription

1 Stem Cells Meeting Report Gene Therapy in Clinical Applications OVERVIEW: How Do We Translate Gene Therapy to Clinical Trials? Curt I. Civin Departments of Oncology and Pediatrics, Johns Hopkins University School of Medicine Hematopoietic gene therapy is a potentially attractive medical tool because of the biology of the lymphohematopoietic stem cell (HSC). The HSC is a favorable cellular target for permanent introduction of genes into the organism, for several reasons, including: Natural and laboratory viruses and potential viral vectors, notably retroviruses, can gain entry to and integrate into the DNA of early hematopoietic progenitor cells. HSC should provide a lifelong source of amplified progeny expressing the introduced gene. The multiple lineages of HSC-derived blood and immune cells traverse the entire organism, and so might correct many inherited enzyme deficiency diseases by secreting enzymes (e.g. storage diseases). Several diseases of blood and immune cells are corrected by successful allogeneic bone marrow transplantation (BMT), and so could potentially be treated by expression of wild type genes in hematopoietic stem cells. Retroviral vectors for hematopoietic gene transfer initially generated many potential concerns, e.g. insertional mutagenesis. Fortunately, most of these concerns have not become clinical problems. Unfortunately, efficacy has been severely limited due to extreme inefficiency of transducing human HSC: Transduction of murine in vitro colony-forming progenitors is much more efficient than transduction of murine transplantable HSC. Transduction of murine in vitro colony-forming progenitors is much more efficient than transduction of human in vitro colony-forming progenitors. Transduction of human in vitro colony-forming progenitors is much more efficient than transduction of human transplantable HSC. There has been considerable recent progress on increasing the efficiency of retroviral transduction. Is hematopoietic gene therapy now close to clinical trials? If so, what are the STEM CELLS 2000;18: next steps toward translating these gains to clinical testing? Some of the next steps toward translating these gains to clinical testing include: Development and utilization of assays that really predict transduction of HSC: Clinical BMT = gold standard In vivo chimera assays (human/fetal sheep, human/immunodeficient mouse) Novel in vitro surrogates? Insuring efficient vector binding to and entry into HSC Pseudotypes; GALV, VSV receptors Insuring efficient vector integration into cellular DNA Lentivirus appears to integrate in resting cells Early acting hematopoietic growth factors appear to recruit HSC into cycle by ex vivo culture before and during transduction with standard RLV Avoiding the silencing of successfully transduced therapeutic gene Insulator sequences which block methylation of the therapeutic gene/promoter Providing specific enhancers for high expression of the successfully transduced therapeutic gene Development of systems for regulated expression that would allow on-off switching in the patient Development of vectors which utilize specific, safe integration sites Avoiding potentially immunogenic and virally infected human proteins Serum-free medium (added dividend: reproducibility!) Recently, Enrico Novelli., Linzhao Cheng, Yandan Yang, and I have focused on the use of hematopoietic growth factor stimulation in serum-free medium to address some of the above problems. We have shown that human cord

2 151 Meeting Report blood CD34 + cells can be transduced with retroviral vectors by ex vivo culture in serum-free medium containing the cytokine combination of stem cell factor, thrombopoiesis, and flt-3 ligand. Transduced cells generate easily detectable human hematopoietic engraftment in immunodeficient mice, and a substantial minority of the human marrow cells in the resulting chimeras contain the transduced marker protein. With Dr. Harry Malech, we are now investigating the use of this system for clinical trials. The Human Sheep Xenograft Model for the Study of the In Vivo Potential of Human HSC and In Utero Gene Transfer Esmail D. Zanjani (summarized by Zhigang Gao and Anita Wokhlu) A modification of the human sheep xenograft model has been developed for the in vivo evaluation of the engraftment and differentiation potential of human hematopoietic stem/progenitor cells. This exhaustion strategy involves the administration of human growth factors to hematopoietic chimeras which have been generated by transplantation of human hematopoietic cells into pre-immune fetal sheep recipients. Initial results indicate that long-term engrafting human stem cells (HSC) can be distinguished from committed progenitor cells by use of this strategy. The model can also be used to assess expansion and transduction of HSC. The late-acting human specific growth factors, GM- CSF and interleukin 3, were administered to human-sheep hematopoietic chimeras on nine days of every month. Administration of these human cytokines was found to augment the levels of human cells in the bone marrow of the chimeras from about 5% to approximately 15%. Hypothetically, these cytokines elevate the levels of human cells in the chimeras by stimulating committed human progenitor cells, potentially exhausting these committed progenitor cells. In contrast, HSC should not be affected by these cytokines. If this is true, persistence of human cells of multiple lineages in chimeras despite this cytokine treatment may be able to be used to assess human HSC. A comparison of the behavior of transplanted human CD34 + CD38 + cell preparations (enriched in committed progenitor cells, depleted of HSC) versus CD34 + CD38 cell preparations (enriched in HSC) tests the utility of this system and hypothesis. In prior published experiments, human CD34 + CD38 cell preparations engrafted after transplantation to primary fetal sheep. Moreover, after the human cells from marrows of the primary sheep were transplanted into secondary fetal sheep hosts, human cells were present persistently in the secondary chimeras. In contrast, CD34 + CD38 + cell preparations engrafted in primary recipients, but not upon secondary transfer. In the exhaustion experiments, chimeras which had been generated by transplantation of CD34 + CD38 + cell preparations were quickly exhausted of their content of human cells. In contrast, chimeras generated by transplantation of CD34 + CD38 cell preparations exhibited delayed exhaustion. Dr. Zanjani reported that the exhaustion strategy can be used to assess the engraftment capability of a population of cells within four to six months. This is much faster than protocols currently used with the human-fetal sheep model to distinguish early human progenitor cells from HSC, which rely on long-term results of serial transplantation experiments. The human-sheep xenotransplantation model was also used to assess the in vivo potential of CD34 + versus CD34 human bone marrow cells. Although both CD34 + and CD34 human bone marrow cells generated persistent engraftment of multiple lineages of human cells in the chimeras, chimeras generated by transplantation of CD34 + cells were exhausted by treatment with four cycles of human growth factors. In contrast, there was not significant exhaustion noted in chimeras generated by transplantation of CD34 cells. Using the exhaustion strategy, the persistence of HSC activity was found to depend on the source of cells, their phenotype, and their concentrations. As illustrated with the CD34 + and CD34 populations, the human sheep xenograft model can be used to further characterize ex vivo expansion, homing, and transduction. The permissive environment of the developing preimmune fetal sheep allowed long-term transfer and expression of Neo -R gene following the direct trans-maternal uterine injection of retroviral vectors into the fetus (i.p.). In utero transplantation of human CD34 + cells followed then by i.p. injection of a high titer retroviral vector resulted in gene transduction of human cells in both primary and secondary chimeric sheep. Of the human cell colonies, 8%-22% expressed Neo -R gene by polymerase chain reaction detection. The levels of transduced cells were comparable in the primary and secondary recipients (up to 11 months after transplantation). Thus, the human-sheep xenograft model provides a biologically relevant assay to determine the in vivo potential of stem cells and may also serve as a pre-clinical model for in utero gene therapy.

3 Meeting Report 152 Transduction of Mouse Hematopoietic Stem Cells is More Efficient with 10A1 Retrovirus Vectors than with Amphotropic Vectors David M. Bodine, Stephane Barrette, Nancy Seidel, Donald Orlic, A. Dusty Miller (summarized by Enrico Novelli and Zhigang Gao) The use of retroviral vectors for gene therapy of human hematopoietic diseases has been hampered by a very low frequency transduction of hematopoietic stem cells (HSC). While it is possible to retrovirally label up to 90% of the murine repopulating HSC, the efficiency of transduction in primates is low, generally <1%. Dr. Bodine noted that a clear difference between these retroviral transductions conducted in mice versus primates lies in the specific vectors employed for the different species. For murine gene therapy, it is possible to use ecotropic retroviral vectors. The ecotropic retroviruses have a GP70 envelope protein which binds to a specific murine cell surface receptor (an amino acid transport protein). To transduce primate cells, amphotropic retroviruses are instead used. Amphotropic retroviruses have a different envelope. The amphotropic envelope protein interacts with a different transport protein receptor, named PiT-2. Dr. Bodine hypothesized that the low number of PiT-2 molecules on primate HSC, as compared to the number of ecotropic receptors on murine HSC, might result in low binding of the amphotropic virus to HSC, and thereby low transduction efficiency. To test this hypothesis, Dr Bodine decided to evaluate mouse HSC for their level of PiT-2 expression. Mouse HSC can be subdivided by elutriation into a FR35Lin c-kit high cell fraction, composed of large size cells with predominantly shortterm engrafting capacity, and FR25Lin c-kit high cell fraction, composed of small cells with long-term engrafting capacity. An average of 30 c-kit high cells can repopulate 100% of recipient mice in murine transplantation studies. The level of ecotropic receptor was high on both cell types by a PCR assay devised in Dr. Bodine s lab. In contrast, the level of amphotropic receptor was lower in both cell types, and PiT-2 levels were higher in the FR35Lin c-kit high fraction cells than in the FR25Lin c-kit high fraction. Thus, according to Dr. Bodine s hypothesis, FR25Lin c-kit high cells are likely to be transduced less efficiently by amphotropic retroviral vectors than the FR35Lin c- kit high fraction cells. To test this, Dr. Bodine exposed both elutriated cell fractions to either ecotropic or amphotropic retrovirus supernatants for 96 h in culture in the presence of interleukin 3 (IL-3), IL-6, and stem cell factor (SCF). He then transplanted the transduced cells into syngeneic recipient mice. Mice were sacrificed 16 weeks post transplantation, and DNA polymerase chain reaction (PCR) analysis of blood from the animals was performed to detect the integrated retrovirus in the progeny of the repopulating HSC. All but one of the mice which received FR25Lin c-kit high cells transduced with the amphotropic retrovirus did not harbor transduced repopulating cells. Conversely, high percentages of the mice that were transplanted with either (A) FR35Lin c-kit high cells transduced with either retroviruses or (B) FR25Lin c-kit high cells transduced with ecotropic retrovirus, contained transduced repopulating cells. This experiment confirmed the hypothesis that the low transduction efficiency observed with the amphotropic retroviruses is not due to low cycling level of the cells but to the low number of PiT-2 receptor molecules on the cell surface. Therefore, Dr. Bodine decided to use two approaches to circumvent this problem. The first is to induce higher levels of PiT-2 expression on HSC by extending the period of in vitro culture. The second is to investigate transduction via PiT-1, the receptor used by Gibbon ape leukemia virus (GaLV). In his talk, Dr. Bodine focused on the results of his experiments aiming to induce higher levels of amphotropic retrovirus on the FR25Lin c-kit high cells, which resemble human HSC. Mouse bone marrow (BM) cells were cultured in IL-3, IL-6, and SCF for up to 192 h, then FR25Lin c-kit high cells were isolated and the mrna levels of PiT-2 and PiT-1 were analyzed by reverse transcriptase-pcr. The levels of both receptors increased during the culture period. The cells were then transduced with amphotropic retrovirus and transplanted into irradiated syngeneic mice. When the cells were cultured for 0-96 h, 5/43 animals were positive for amphotropic proviral sequences, versus 7/15 animals transplanted with cells precultured for 144 h. Dr. Bodine calculated that at least six days of preculture are necessary to enhance the levels of PiT-2 to allow efficient gene transfer. Dr. Bodine drew the conclusion that the high levels of PiT-2 mrna obtained after 144 h of preculture led to improved transduction with amphotropic retroviral vectors. However, this study raised the concern that the extended preculture period might have partially depleted the HSC capacity of the cultured cells. A competitive repopulation assay demonstrated that mouse BM cells cultured for eight days in the same growth factors retained 60% of their initial repopulating ability. The future direction of Dr. Bodine s lab aims at developing strategies to induce the amphotropic receptor levels on murine and human HSC without depleting their repopulating capacity. Strategies may include the use of serum-free medium, different combinations of cytokines, or mobilization of HSC with a cytokine combination which

4 153 Meeting Report leads to the induction of high levels of amphotropic and GaLV receptors on mobilized peripheral blood CD34 + CD38 cells. Finally, in collaboration with Donald Orlic, Dr. Bodine showed that cryopreserved human cord blood (CB) cells have much higher levels of amphotropic receptor than freshly isolated CB cells. This seems to correlate with a higher transduction efficiency; when transduced cells were tested for the ability to repopulate SCID mice, highly transduced cells of multiple lineages were recovered from the animals. Dr. Bodine will focus on elucidating the mechanisms that underlie the higher amphotropic receptor expression levels in cryopreserved HSC. Unrelated Placental Blood in Marrow Transplantation J. Kurtzberg, P. Martin, N. Chao, C. Stevens, P. Rubinstein (summarized by Margaret Stull and Rachata Lumkul) The use of umbilical cord blood (UCB) for hematopoietic stem cell transplantation has recently received great attention due to the possibility of increased engraftment potential and decreased incidence of graft versus host disease (GVHD) for UCB, as compared to bone marrow (BM). Indeed, several studies have suggested that UCB may be an enriched source of hematopoietic stem cells. In 1992, the New York Blood Center initiated the Placental Blood Project, through which UCB units are collected from healthy newborns at Mt. Sinai Hospital. The phase I trial using the banked UCB for transplant grafts began five years ago as a collaboration between Dr. Kurtzberg s group at Duke University and Dr. John Wagner s group at The University of Minnesota. In this trial, researchers are attempting to determine the efficacy of banked partially mismatched UCB in hematopoietic stem cell transplants. Concerns in performing unrelated UCB transplants include the number of cells that can be harvested and the feasibility of using this procedure in adults, the limits of HLA mismatch, GVHD incidence and severity, and graft versus leukemia (GVL) effects. The 167 consecutively transplanted patients included in Kurtzberg s report suffered from hematological malignancies, congenital and acquired bone marrow failure conditions, and genetic diseases. They ranged from 0.4 to 58 years of age, with the median age being 7.1 years. At the time this information was presented, data were updated through October 1998, and all patients had been observed following transplant for at least 100 days and up to 66 months. In these UCB transplants, UCB cell dose is typically 10-fold lower than that given with BM, averaging approximately nucleated cells/kg, with CD34 + cells/kg. Infants may receive a cell dose equal to that given with BM ( nucleated cells/kg). Of the 167 patients, 124 were treated at Duke and 43 at Minnesota; some differences exist between the two centers approaches. All patients at Minnesota received total body irradiation (TBI), whereas only one-half of the Duke patients had TBI; patients at Minnesota had a lower dose of steroid regimen with cyclosporin than one-half of those at Duke. Researchers at Duke gave more mismatched grafts than those at Minnesota, so they have a greater number of patients with <5/6 HLA match, and only 8% of their patients received a 6/6 match. Similarities include the provision of standard supportive care to all patients and the administration of G-CSF, IVIG, GVHD prophylaxis. In both centers, the preparative regimen was busulfan and cytoxan for all patients with nonneoplastic diseases. Duke patients with malignancies received melphalan with TBI and/or busulfan while Minnesota patients received cyclophosphamide. The matching strategy used by Dr. Kurtzberg s group has evolved during this study. Initially, matching was prioritized in the following order: best HLA matched unit, Class I serology, and DRβ-1. Later, data from BM transplants showed the advantage of DRβ-1 matching over Class I serology. Currently, cell dose is prioritized over DRβ-1 and Class I serology matching. In other words, the researchers would select a larger cell number (dose) with 4/6 HLA match and DRβ-1 match over a smaller total cell number with 6/6 HLA match. Results show 93% of the patients engrafted eventually. The engraftment, measured as the achievement of an absolute neutrophil count (ANC) of 500, was found to be correlated with the number of CD34 + cells transplanted, rather than the degree of HLA matching. In spite of the finding of Kurtzberg s group that cell dose overrides the HLA disparity, this effect may be due to the fact that there are few 6/6 matches in this study. Nonetheless, their study shows that a dose of CD34 + cells < CD34 + cells/kg results in diminished and delayed engraftment of platelets and neutrophils. Multivariate analysis shows that any dose > CD34 + cells/kg results in fairly consistent engraftment, which has led the Kurtzberg group to consider a dose of CD34 + cells/kg the lower threshold for a positively engrafting transplant. Patients who received G-CSF had a nine-day advantage in achieving an ANC of 500 over those who did not receive G-CSF; G-CSF had no affect on platelet engraftment or overall survival.

5 Meeting Report 154 Forty percent of patients experienced levels II-IV GVHD, while 14% reached levels III-IV. These proportions are less than what would be expected from a comparable (historical) transplant with BM, and even those with levels III-IV GVHD following UCB transplant had a higher percent survival than patients with levels III-IV GVHD following historical BM transplant. Additionally, there was no difference in acute GVHD between patients with 1, 2, or 3/6 HLA mismatching. The occurrence of chronic GVHD was low (11%) compared to the historical incidence after BM transplant (70%) and was limited, rather than extensive, in severity. GVHD prophylaxis continued for 9-12 months after transplant. Patients were given steroids for the first two to three months and cyclosporin for the first 9-12 months, and then they came off prophylaxis completely. Phytohemagglutinin and lymphocyte counts were normal and remain normal after this period, and blood levels of CD45RA T lymphocytes remained elevated for up to three to four years. Some transplants failed due to non-relapse mortality, which occurred in the first three months; half of these cases were due to viral, bacterial, and sepsis infections. Adults tended to suffer more infections than children, and there was a lag of approximately one year to reaching normal CD4 + counts in adults as compared to children. Kurtzberg s group has also performed expansion studies using the Aastrom system, which is designed for BM cell expansion. Twenty-eight patients were given a portion of UCB cells on day 0 by a conventional transplant and were later boosted on day 12 with cells that had undergone expansion in Aastrom media containing FLT-3, PIXY, low dose erythropoietin (EPO), fetal calf and horse serum. Following expansion, there was a 4- to 10-fold increase in myeloid cells and a loss of lymphoid cells. In the patients, there was no advantage to myeloid, erythroid, or platelet engraftment, although a positive relationship was found between colony-forming unit-granulocyte/macrophage dose and platelet engraftment. The Food and Drug Administration (FDA) has approved a randomized trial in which patients will receive either fresh or expanded/cultured UCB, but there will not be enough units banked to begin this experiment for one to two years. In the meantime, Kurtzberg s group continues to experiment with culture conditions. The standard Aastrom expansion media contains interleukin 3/GM-CSF, EPO, FLT-3, and stem cell factor (SCF). Current government regulations do not permit the use of SCF in clinical trials, so the Kurtzberg lab has instead made placental adherent cells from placental tissue that was frozen simultaneously with UCB. Without SCF but with placental stroma or BM stroma, Kurtzberg has found an increase in expansion when compared to Aastrom conditions. With the addition of SCF to the media, expansion increases even further. If SCF is not soon approved by the FDA for clinical use, Kurtzberg plans to obtain BM stromal cells from the patient before UCB transplant, expand UCB cells on the BM cell stroma, and harvest them together for the transplant. Role of Serum-Free Medium in the Ex Vivo Expansion of Human Cord Blood Hematopoietic Stem Cells Richard K. Shadduck, Gary L. Gilmore, John Lister (summarized by Vivek Tanavde) Dr. Richard Shadduck presented recent results on ex vivo expansion of cord blood stem/progenitor cells. He stressed the importance of using early acting cytokines like flt-3 ligand (FL) and thrombopoietin (TPO) in the ex vivo culture medium. Using conventional cytokines such as G-CSF, GM-CSF, and interleukin 3 (IL-3), other labs are able to achieve only limited expansion (two- to threefold) of cord blood CD34 + cells in vitro as quantified by flow cytometry. However using culture medium containing FL and TPO [1], the Shadduck lab was able to achieve much more impressive ex vivo expansion (20- to 30-fold) of cord blood CD34 + cells. Using this system, enough CD34 + cells could be generated from a single collection for potential engraftment of an adult (based on numbers of CD34 + cells/kg known to be required for engraftment). Addition of stem cell factor and IL-6 to the system further enhanced the numbers of CD34 + cells by two- to fourfold. Thus, 60- to 100-fold expansion of CD34 + cells from cord blood was observed by four to six weeks ex vivo culture. They further studied the kinetics of stem cell expansion using this culture system. Using total cell count as a parameter in this study, a lag period of four to five weeks was observed when whole cord blood was used for expansion. A plateau in the growth curve was observed after four to five weeks when whole cord blood mononuclear cells were used. The lag was reduced to one week when purified CD34 + cells from cord blood were used. The frequency of the more primitive CD34 + /CD38 dim population increased 10-fold in two months of culture using the same culture system. Using serum free medium (QBSF-60) containing the above-mentioned cytokines (FL + TPO) for culture of CD34 + cells isolated from cord blood, the Shadduck lab observed that the cell numbers did not increase in the absence of serum. However, if cord blood CD34 + cells were cultured in

6 155 Meeting Report serum-containing medium for an initial period of one to two weeks, the cultured cells could then be successfully transferred to QBSF-60 medium and expanded. Therefore, it appears that serum contains some uncharacterized growth factor(s) required for initiation of the CD34 + cell expansion in culture. Thus, this ex vivo culture system has potential for clinical application for adult patients needing allogeneic stem cell transplants; ex vivo culture might be able to expand CD34 + cell numbers sufficiently to allow cord blood transplantation of large adult recipients. The Shadduck lab has recently developed a system using teflon bags for ex vivo culture of stem cells, and they are planning to perform long-term culture-initiating cell assays and in vivo engraftment assays to further evaluate potential stem/progenitor cell expansion in this system. Up to now, their emphasis has been on immunophenotyping of the ex vivo cultures without functional assay data. If encouraging results are obtained, cells could be transplanted into the patients directly from these cultures done in a closed system with minimum manipulation of the cells. The Shadduck lab is also attempting to identify the serum-derived factor(s) needed to enable initiation of primary cultures of cord blood CD34 + cells in serum-free medium. REFERENCES 1 Piacibello W, Sanavio F, Garetto L et al. Extensive amplification and self-renewal of human primitive hematopoietic stem cells from cord blood. Blood 1997;89: Use of Serum-Free Medium with Fibronectin Fragment Enhanced Transduction in a System of Gas Permeable Plastic Containers to Achieve High Levels of Retrovirus Transduction at Clinical Scale Harry L. Malech (summarized by Rachata Lumkul and Vivek Tanavde) Chronic granulomatous disease (CGD) is an inherited immunodeficiency disease which occurs in about five per million humans. The disease presents clinically with fungal and bacterial infections in multiple organs. In affected patients, neutrophils, monocytes, and eosinophils fail to destroy microbes utilizing SO and H 2 O 2. Now that the relevant genes have been cloned, it is clear that there are four clinical/genetic subtypes of CGD. Each mutation affects a different chain of the phagocyte oxidase (phox) microbiocidal enzyme. It is likely that even low efficiency gene therapy may have a therapeutic effect in CGD patients because granulocytes from some clinically normal female carriers of X-linked CGD have only 3%-5% of the normal phox enzymatic activity. With this rationale, Dr. Malech developed clinical scale methods for ex vivo transduction of mobilized peripheral blood CD34 + cells for gene therapy trials in CGD patients. In this symposium, he described the development and application of stem cell culture and retroviral transduction protocols using a closed system (gas-permeable plastic bloodbags), serum-free medium containing recombinant human hematopoietic growth factors, and fibronectin fragment-augmented retroviral transduction. In Dr. Malech s phase I clinical trial, five patients with autosomal recessive p47 phox deficient CGD received intravenous infusion of autologous CD34 + peripheral blood stem cells (PBSC) which had been transduced ex vivo with a recombinant retrovirus encoding normal p47 phox (amphotropic MFGS-p47 phox retroviral vector). He used the Isolex 300 stem cell selection system (Nexell; Irvine, CA) to purify CD34 + cells from the leukapheresis cell product from G- CSF mobilized patients. Retroviral vector was harvested from producer lines cultured overnight in serum-free medium (BioWhittaker; Walkersville, MD; X-vivo 10 TM with 1% human serum albumin). The CD34 + cells were cultured and transduced in gas permeable flexible plastic containers (bloodbags) designed for stem cell culture (Nexell PL2417). The CD34 + cells were transduced twice, on days 2 and 3 of culture in serum-free medium containing recombinant human growth factor (interleukin 3 [IL-3]/GM-CSF fusion protein). Then the cells were washed and resuspended in plasmalyte containing 1% human serum albumin for Inheritance Gene mutated Human chromosomal localization Percent of CGD cases X-linked gp91 phox X 60 Autosomal recessive p47 phox 7 30 Autosomal recessive p67 phox 1 2 Autosomal recessive p22 phox 16 2

7 Meeting Report 156 intravenous administration to the patient. The patients did not receive any conditioning regimen. Dr. Malech analyzed the transduction of patient blood cells and correction of phagocyte oxidase activity by using a phorbol 12-myristate 13-acetate (PMA)-stimulated nitroblue tetrazolium dye (NBT) test. A flow cytometry assay of oxidant production using dihydrorhodamine 123 (DHR) loading of the cells also was used to detect NADPH oxidase positive neutrophils in peripheral blood of patients after gene therapy. The results of this first clinical trial detected phox + granulocytes in peripheral blood of all five patients. Peak levels of corrected cells occured three to six weeks after infusion, and ranged from 0.004%-0.05% of total peripheral blood granulocytes. The %NBT + colonies was between 9%-30%. Corrected cells were detectable for as long as six months after infusion, in some individuals. This trial also demonstrated the successful use of animal protein-free medium and a blood-bank-compatible closed system of gas permeable plastic containers for culture and transduction of PBSC. In his second clinical trial, Dr. Malech studied gene therapy in the most common and severe form of CGD, X-linked gp91 phox deficiency. For each cycle of gene therapy, patients were given eight daily subcutaneous injections with the combination of 50 µg/kg flt-3 ligand and 5 µg/kg GM-CSF to mobilize CD34 + cells. Apheresis was performed when mobilization peaked at days CD34 + cells were selected from leukapheresis products using the Isolex 300 (Nexell) which resulted in 80%-90% pure CD34 + cells at 60%-70% yield. Selected CD34 + cells were cultured and transduced in X-vivo TM 10 supplemented with 1% human serum albumin and growth factors (IL-3/GM-CSF fusion protein plus Flt-3 ligand). Dr. Malech prepared a high titer amphotropic MFGS-gp91 phox retrovirus vector. Culture and transduction were done in PL2417 containers coated with CH296 (Retronectin, Takara-Shuzo Corp.; Kyoto, Japan). CD34 + cells were exposed to serum -free retrovirus vector for seven h daily for four days, resulting in final transduction ranging from 48%-89% of colony-forming cells. By the end of this culture plus transduction, >70% of cells remained CD34 +, and there was a >3-fold expansion of total cell numbers. Trial design and analysis were similar to the first trial. Three male patients with X-linked CGD each received two cycles of gene therapy without bone marrow conditioning; cycles were 50 days apart. In two patients, peak numbers of phox + neutrophils were detected in peripheral blood (by the DHR flow cytometry assay) at three to four weeks after each cycle of gene therapy (1 in 500-1,700 cells; 0.2%-0.6%). In one patient receiving treatment for a fungal liver abscess, phox + neutrophils were detected in pus draining from the abscess at four months after the first treatment. Two patients had >1 in 4,000 phox + neutrophils in their peripheral blood. Thus, these improvements from the first trial resulted in a marked increase in ex vivo transduction efficiency. In one patient, peak levels of circulating phox + neutrophils were >4-fold higher than the best results of the first trial. In conclusion, Dr. Malech reports successful retroviral transduction in a clinical trial. Nevertheless, the current trials have achieved only low level, relatively short-term expression of the introduced gene. Further enhancements of the levels of stem cell transduction are anticipated in planned trials using additional hematopoietic growth factors (e.g., SCF, thrombopoietin, Flt-3 ligand) and/or alternative vectors (e.g., lentivirus).