[Show abstract][Hide abstract] ABSTRACT: Although novel retroviral vectors for use in gene-therapy products are reducing the potential for formation of replication-competent retrovirus (RCR), it remains crucial to screen products for RCR for both research and clinical purposes. For clinical-grade gammaretrovirus-based vectors, RCR screening is achieved by an extended S(+)L(-) or marker-rescue assay, whereas standard methods for replication-competent lentivirus detection are still in development. In this report, we describe a rapid and sensitive method for replication-competent gammaretrovirus detection. We used this assay to detect three members of the gammaretrovirus family and compared the sensitivity of our assay with well-established methods for retrovirus detection, including the extended S(+)L(-) assay. Results presented here demonstrate that this assay should be useful for gene-therapy product testing.Gene Therapy advance online publication, 8 March 2012; doi:10.1038/gt.2012.18.
[Show abstract][Hide abstract] ABSTRACT: A total of 50 consecutive patients (median age, 57.5 years) with AML (n=30) or myelodysplasia (MDS, n=20) underwent HLA matched related donor (MRD, n=27) or unrelated donor (MUD, n=23) peripheral blood hematopoietic cell transplantation after nonmyeloablative CY/fludarabine (Flu) conditioning. GVHD prophylaxis included CsA (n=19)+/-mycophenolate mofetil (n=31). At a median follow-up of 59 months, 21 patients (42%) were alive without evidence of disease. By Kaplan-Meier analysis, year 1-4 disease-free survival (DFS) and OS estimates were 0.50/0.58, 0.40/0.46, 0.37/0.43 and 0.37/0.41. MUD recipients were engrafted quickly (13.5 days) compared to MRD recipients (16 days) and relapsed/progressed less frequently (P=0.005). Overall grade 3/4 acute GVHD (aGVHD) occurred in 26% in the absence of antecedent mucositis and was associated with chronic GVHD (cGVHD) and poor OS. Extensive cGVHD developed in 51.2% of 100 day survivors. Rates of aGVHD, cGVHD and survival were similar between MRD and MUD recipients. Of 14 survivors with cGVHD, 5 (35.7%) experienced resolution off immunosuppression, suggesting that tolerance with HLA matched grafts is possible at an advanced age by this method. This study provides further evidence for prolonged DFS after CY/Flu MRD allotransplantation for AML/MDS, and extends the findings to older patients and those with unrelated donors.
Bone marrow transplantation 08/2010; 45(8):1300-8. · 3.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Research in gene therapy involving genome-integrating vectors now often includes analysis of vector integration sites across the genome using methods such as ligation-mediated PCR (LM-PCR) or linear amplification-mediated PCR (LAM-PCR). To help researchers analyze these sites and the functions of nearby genes, we have developed SeqMap (http://seqmap.compbio.iupui.edu/) a secure, web-based comprehensive vector integration site management tool that automatically analyzes and annotates large numbers of vector integration sites derived from LM-PCR experiments in human and model organisms upon a common genome database. We believe the use of this resource will enable better reproducibility and understanding of this important data.
[Show abstract][Hide abstract] ABSTRACT: Administration of chemotherapy is often limited by myelosuppression. Expression of drug-resistance genes in hematopoietic cells has been proposed as a means to decrease the toxicity of cytotoxic agents. In this pilot study, we utilized a retroviral vector expressing methylguanine DNA methyltransferase (MGMT) to transduce hematopoietic progenitors, which were subsequently used in the setting of alkylator therapy (procarbazine, CCNU, vincristine (PCV)) for poor prognosis brain tumors. Granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells were collected by apheresis and enriched for CD34+ expression. Nine subjects were infused with CD34+-enriched cells treated in a transduction procedure involving a 4-day exposure to cytokines with vector exposure on days 3 and 4. No major adverse event was related to the gene therapy procedure. Importantly, the engraftment kinetics of the treated product was similar to unmanipulated peripheral blood stem cells, suggesting that the ex vivo manipulation did not significantly reduce engrafting progenitor cell function. Gene-transduced cells were detected in all subjects. Although the level and duration was limited, patients receiving cells transduced using fibronectin 'preloaded' with virus supernatant appeared to show improved in vivo marking frequency. These findings demonstrate the feasibility and safety of utilizing MGMT-transduced CD34+ peripheral blood progenitor cells in the setting of chemotherapy.
Cancer Gene Therapy 10/2006; 13(9):886-95. · 2.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The National Gene Vector Laboratory (NGVL) is a US National Institutes of Health initiative charged with providing clinical grade vectors for gene therapy trials. The program was started in 1995 and Indiana University has served as the production site for retroviral vectors and is also accepting applications for production of lentiviral vectors. The facility is designed to produce vectors for Phase I and Phase II clinical trials with the specific mandate to facilitate investigator-initiated research for academic institutions. To date, the facility has generated over 30 Master Cell Banks for gene therapy investigators throughout the United States. This required the facility to develop a system that can adapt to the varied needs of investigators, most of whom request different vector backbones, packaging cell lines, final product volumes, and media. In this review, we will illustrate some of the experiences of the Indiana University NGVL during the generation of retroviral vectors using murine-based packaging cell lines.
[Show abstract][Hide abstract] ABSTRACT: To better characterize lentiviral vector supernatants, we compared three methods of titer assessment. These titer methods include assessment of vector RNA sequences in supernatants, DNA sequences in transduced cells, and vector expression in transduced cells (using a vector which expressed the green fluorescence protein, GFP). For analysis of RNA and DNA, we developed a real-time PCR method for detecting the lentiviral packaging sequence and used this methodology to quantitate the number of vector sequences. Vector expression was assessed by flow cytometric analysis for GFP. As functional titers (DNA and GFP expression titers) are dependent on transduction efficiency, we calculated the titer of a lentiviral vector, RRL-CMV-GFP, after transduction of 293, HeLa, or Mus dunni cells. Genomic DNA was extracted at 4 and 14 days after transduction and the number of vector DNA molecules was determined against a plasmid standard. Of the three cell lines tested, 293 cells provided the highest rate of transduction (PCR estimated DNA titer for RRL-CMV-GFP vector was 2.52 +/- 0.25 x 10(6) molecules/ml at 14 days, and 2.31 +/- 0.15 x 10(6) molecules/ml at 4 days). When titer was calculated based on GFP expression, the highest titer was also obtained on 293 cells (0.26 +/- 0.04 x 10(6) TU/ml at 14 days, and 0.24 +/- 0.03 +/- 10(6) TU/ml at 4 days). The titers obtained by GFP expression assay were approximately one log lower than those obtained by DNA analysis suggesting that variability in vector expression may underestimate titer. Measurement of RNA titers directly from vector supernatants against a plasmid standard indicated that the RNA titers are substantially higher than the DNA (approximately 10(3)-fold) and GFP titers (approximately 10(4)-fold). To show that the lentiviral probe and primers could be used for titering a variety of lentiviral vectors, we have also used the real-time PCR method to determine the DNA titers of two other HIV1 derived vectors, RRL-PGK-GFP (6.1 +/- 1.4 x 10(5) molecules/ml), and SMPU-RRE-BN (1.26 +/- 0.2 x 10(6) molecules/ml). We conclude that of the three methods tested, titers assessed by DNA analysis of transduced cells provide the most reliable estimate of functional titers as these are least likely to be influenced by factors, such as defective interfering particles and vector expression levels. The real-time PCR method described offers a reproducible method for lentiviral titering and can be applied to a wide variety of vectors, regardless of transgene.
[Show abstract][Hide abstract] ABSTRACT: Thymoma is a chemotherapy-sensitive tumor with a 30-50% 5-year survival in previously untreated patients. Unfortunately, durable CRs with salvage chemotherapy are rarely observed. We initiated a phase II trial of high-dose carboplatin and etoposide in patients with relapsed thymoma or thymic carcinoma. All patients had progressive disease (PD) after initial or salvage chemotherapy, but were not cisplatin-refractory. PBSCs were mobilized using 10 microg/kg/day G-CSF. Patients received carboplatin 700 mg/m(2) and etoposide 750 mg/m(2) i.v. on days -5, -4, -3. Five patients were enrolled and evaluated after tandem transplants 4 weeks apart. All patients had pleural-based and lung parenchymal metastasis, one or two prior surgeries and two or more courses of prior cisplatin-based chemotherapy regimens. Chemotherapy was well tolerated, although grade IV hematological toxicity occurred in all patients. Progression-free survival following HDC ranged from 3.5 to 16.5 months. One patient maintained a CR for 12.8 months, then died from an unrelated cause. With a minimum of 2 years follow-up for all patients, three of five patients remain alive at 26+, 36+, and 49+ months. High-dose carboplatin and etoposide in relapsed thymoma is feasible with acceptable toxicity; however, these limited data do not appear superior to standard-dose salvage therapy.
Bone Marrow Transplantation 10/2001; 28(5):435-8. · 3.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There are many studies that examine the psychosocial adjustment of survivors of bone marrow transplantation (BMT). On the other hand, there are relatively few studies that examine the psychosocial adjustment of patients prior to BMT, and even fewer that focus on the psychosocial adjustment of the patient's caregiver. The purpose of the present study was to assess performance status and psychosocial adjustment to illness, mood and stress response of patients and caregivers prior to admission for allogeneic BMT. Forty patients and their 39 caregivers were assessed using standardized measures. One-fourth of the patients reported clinical levels of psychosocial maladjustment on the Psychosocial Adjustment to Illness Scale and had greater adjustment problems than BMT survivors. Approximately one-third (35%) and one-quarter (23%) of the patients reported significant symptoms of intrusive and avoidance stress responses, respectively on the Impact of Events Scale. Caregivers reported more impairments in family relationships than patients, but overall reported similar distress to that of patients. Information about the pre-BMT process appears to be critical to understanding the psychosocial impact that BMT can have on patients and their caregivers.
Bone Marrow Transplantation 07/2001; 27(11):1181-8. · 3.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Investigators conducting retroviral gene therapy trials are required to monitor for the presence of replication-competent retrovirus (RCR). The required testing utilizes a combination of biologic assays and molecular tests using PCR. In the course of a human clinical gene therapy trial, we detected 4070A viral envelope sequences in CD34(+) peripheral blood stem cells 2 days after transduction using a PCR-based assay, suggesting the presence of RCR. The supernatant and producer cells used for vector generation had been negative in extensive screening using the extended S(+)/L(-) assay. The presence of a replication-competent virus was subsequently excluded by a combination of biologic and PCR analyses. The source of the 4070A viral envelope sequences was determined to be packaging cell line DNA in the vector supernatant. The analysis of a variety of vector supernatants by quantitative real-time PCR revealed 4070A envelope DNA sequences from the packaging cell line in concentrations equivalent to approximately 50-500 focus-forming units per milliliter of wild-type 4070A virus. When PCR was performed after reverse transcriptase treatment of supernatant (i.e., assessing both RNA and DNA content), 4070A envelope sequence concentrations ranged from 10(2) to 3.5 x 10(3) focus-forming units per milliliter of wild-type 4070A virus. Our data indicate that PCR should not be used to analyze transduced cells for RCR within the first 2 weeks of vector exposure. Furthermore, investigators using PCR to analyze transduction efficiency shortly after vector exposure may experience false-positive findings.
[Show abstract][Hide abstract] ABSTRACT: The potential pathogenicity of replication-competent retroviruses (RCR) requires vigilant testing to exclude inadvertent contamination of clinical gene therapy vector products with RCR. Pseudotyped vectors using the gibbon ape leukemia virus (GALV) envelope have entered into clinical trials but specific recommendations regarding methods for screening of vector product and analysis of clinical samples have not been set forth. Unfortunately, current screening assays used for detecting amphotropic RCR are not suitable for GALV-pseudotyped RCR. We modified the extended S+/L- assay for RCR detection by using human 293 cells for virus amplification. Of five cell lines tested, 293 cells were selected because they combined a high transduction efficiency and an ability to generate RCR at high titer. After optimizing the amplification assay, a dilution of GALV virus could consistently be detected at a dilution of 10(-6). In coculture experiments, one GALV-infected cell could be consistently detected in 10(6) uninfected cells. A PCR-based assay was developed that was capable of detecting 100 copies of a GALV envelope containing plasmid diluted in 1 microg of DNA obtained from uninfected cells. PCR was also able to detect one GALV-infected cell in 10(6) uninfected cells. These assays will be suitable for testing of vector preparations and for monitoring of clinical samples from patients treated in clinical gene therapy protocols. The assays developed are similar in methodology and sensitivity to those currently used for certification of amphotropic retroviral vectors.
Human Gene Therapy 02/2001; 12(1):61-70. · 4.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Production of retroviral vectors for clinical use requires removal of cells and cellular debris. We combined a series of filters of decreasing pore size using commercially available blood banking filters approved for clinical use. The collection bag and filters can be connected to create a sterile, closed system using clinically approved tubing and sealing systems. Even when challenged with a large number of vector producer cells (2.38 x 10(9)cells), no viable cells are passed through the system. The step filtration system developed minimizes the titer reduction associated with filtration, provides rapid flow rates, and was cost effective when filtering volumes in excess of 2 liters.
[Show abstract][Hide abstract] ABSTRACT: We describe collection and purification of peripheral blood CD34+ cells from volunteer, normal donors and allogeneic stem cell donors. A total of 98 aphereses were performed on 68 volunteer donors using peripheral venous access. The mean number of nucleated cells collected was 4.6 x 10(10) which included 1.9 x 10(8) CD34+ cells corresponding to 2.7 x 10(6) CD34+ cells/kg. The number of CD34+ cells collected did not differ between males and females but did correlate with the donor's weight and the total number of nucleated cells collected. The Nexell Isolex 300i cell separator was used to isolate CD34+ cells from 30 of the collections. A mean of 0.36% of the total cells was recovered and included 43 +/- 18% of the CD34+ cells. CD34+ cells represented 85 +/- 11% of the recovered cells. The total number of CD34+ cells recovered was not influenced by the number of nucleated cells placed on the Isolex 300i. The percentage of CD34+ cells recovered was not related to the number of CD34+ cells placed on the Isolex 300i. The purity of the final product was influenced by the number of CD34+ cells but not the total number of nucleated cells. An additional 38 CD34+ cell isolations were performed on normal allogeneic stem cell donors with similar results. These observations further support the safety and feasibility of peripheral blood CD34+ cell collection and purification.
Bone Marrow Transplantation 01/2001; 26(12):1271-9. · 3.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To assess the role of high-dose chemotherapy as initial salvage chemotherapy in patients with relapsed testicular cancer.
From August 1992 to April 1998, 65 patients with testicular cancer were treated with high-dose carboplatin and etoposide followed by peripheral-blood stem-cell transplantation or autologous bone marrow transplantation rescue as initial salvage chemotherapy at Indiana University. An identical course was given after hematopoietic reconstitution. Postchemotherapy resection of residual disease was performed in selected patients with incomplete radiographic response associated with normalization of markers. The median follow-up was 39 months (range, 16 to 91 months).
Thirty-seven (57%) of the 65 patients are continuously disease-free. Three additional patients are disease-free with subsequent surgery. High-dose chemotherapy was associated with significant morbidity but no treatment-related mortality.
High-dose chemotherapy as initial salvage chemotherapy achieved impressive long-term survival with acceptable toxicity in patients with relapsed testicular cancer.
Journal of Clinical Oncology 11/2000; 18(19):3346-51. · 18.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During the production of clinical-grade retroviral vector supernatant, we noted significant differences in the lactate production and glucose consumption of various producer cell lines submitted to the National Gene Vector Laboratory (NGVL). Since differences in growth characteristics could be important in determining the optimal culture conditions for maximizing titer, we studied the growth characteristics of three commonly used packaging cell lines: PA317, PG13 and GP+envAM12. A transformed phenotype, assessed by the ability to form colonies in semisolid media, was evident in all three packaging cell lines tested. In confluent cultures, the rates of glucose consumption and lactate production (per cell per hour) were similar for the three lines tested, but the growth rate and culture density varied. PA317 and PG13 continued to expand after reaching confluence, resulting in higher cell densities and subsequent rapid depletion of glucose within the 24-hr observation period. When the cell lines were evaluated for titer optimization, the slower growing packaging cell line GP+envAM12 generally provided the highest titer after 8 hr of culture in confluent roller bottles, while most vectors introduced into PA317 and PG13 cells yielded optimal titers after 24 hr of culture. We also found that the improved titers obtained by culturing cells at 32 degrees C previously reported for PA317 cells do not apply to other packaging cell lines. In particular, PG13 rapidly lost titer when grown at the lower temperature. Our findings suggest that optimization of titer requires careful consideration of the culture conditions, which should be individualized for the vector producer cell line.
Human Gene Therapy 11/2000; 11(15):2093-103. · 4.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pre-clinical studies indicate that efficient retrovirus-mediated gene transfer into hematopoietic stem cells and progenitor cells can be achieved by co-localizing retroviral particles and target cells on specific adhesion domains of fibronectin. In this pilot study, we used this technique to transfer the human multidrug resistance 1 gene into stem and progenitor cells of patients with germ cell tumors undergoing autologous transplantation. There was efficient gene transfer into stem and progenitor cells in the presence of recombinant fibronectin fragment CH-296. The infusion of these cells was associated with no harmful effects and led to prompt hematopoietic recovery. There was in vivo vector expression, but it may have been limited by the high rate of aberrant splicing of the multidrug resistance 1 gene in the vector. Gene marking has persisted more than a year at levels higher than previously reported in humans.
Nature Medicine 07/2000; 6(6):652-8. · 22.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tumor contamination of autologous peripheral blood stem/progenitor cell grafts occurs in a substantial proportion of high-risk breast cancer patients, and the possibility that such contamination may contribute to relapse has focused attention on methods for removal of the contaminating cells prior to transplantation. One such approach is positive selection of CD34+ cells. A fully automated immunomagnetic cell selection system has recently been introduced to facilitate the positive selection process. A multicenter randomized clinical trial was designed to evaluate the capacity of CD34+ cells isolated using the fully automated system to support prompt hematopoietic reconstitution following high-dose chemotherapy in high-risk breast cancer patients, as well as to assess the safety and tolerability of the CD34+ cell transplants. In recipients of isolated CD34+ cells, the median time to an absolute neutrophil count > or =500/microl was 10 days, a value identical to that observed in patients receiving unfractionated apheresis collections. In the isolated CD34+ cell recipients median time to a platelet count > or =20 000/microl was 12 days, compared with 10 days in the unfractionated cell group. There were no statistically significant differences between the groups in median time to neutrophil or platelet engraftment. Infusion of autologous cells was well tolerated by the study groups. There were no inter-group differences in the incidence of infections, need for platelet transfusions, or duration of hospitalization. Isolated CD34+ cells were high in purity and sufficient in number for use in autologous transplantation. The fully automated immunomagnetic cell selection system affords an efficient and time-saving option for isolation of CD34+cells to be used as autologous grafts in high-risk breast cancer patients, and the isolated CD34+ cells support undelayed hematopoietic reconstitution.
Bone Marrow Transplantation 06/2000; 25(11):1165-74. · 3.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Flt3-ligand (Flt3-L) is an early acting costimulatory cytokine that has been shown to possess antitumor properties in murine solid tumor models. Flt3-L is a trans-membrane protein (tm) but can be proteolytically cleaved to a soluble form, which is also biologically active. In this study, the antitumor effect of both soluble and tmFlt3-L was evaluated in a mouse leukemia model. To mimic the multiorgan involvement characteristic of human leukemia, a factor-dependent cell line FDC.P1 was made leukemogenic by transfection with the human BCR/ABL gene. The resulting cell line, AW, expresses BCR/ABL RNA and protein. It maintains a similar in vitro growth rate as the parent cell line, but unlike the parent cell line, AW cells are factor independent and tumorigenic. Growth of FDC.P1 and AW cells are unaffected by the addition of soluble human Flt3-L to the culture medium. Also, AW growth is unaltered after transduction with a retroviral vector expressing the tm isoform of human Flt3-L (AW/tmFlt3-L). When 10(5) AW cells were i.v. injected into syngeneic DBA/2 mice, fatal leukemia developed in nine of nine (100%) mice within 4-6 weeks with involvement of the blood, bone marrow, spleen, and thymus. Systematic administration of soluble human Flt3-L (500 microg/kg/day) for 10 days protected mice from leukemia, with 11 of 17 mice tumor free at week 8 (64.7%) The tm isoform of Flt3-L also was protective. When 10(4) AW/tmFlt3-L cells were injected i.v. into mice, only 35.7% (5 of 14) developed leukemia versus 100% in control groups. Adoptive transfer of immunity was also demonstrated; T cells obtained from tumor-free animals conferred protection to 87% (seven of eight) naive mice challenged with AW cells. These results demonstrate that both soluble and membrane-bound human Flt3-L has antitumor activity in this leukemia model.
Cancer Research 05/2000; 60(7):1895-900. · 8.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CK beta-11 chemoattracts T cells, B cells, dendritic cells, macrophage progenitors, and NK cells and facilitates dendritic cell and T cell interactions in secondary lymphoid tissues. We hypothesized that expression of CK beta-11 in tumor cells may generate antitumor immunity through these interactions. After transduction with the retroviral vector L(CK beta 11)SN, the murine breast cancer cell line C3L5 (C3L5-CK beta 11) showed expression of retroviral mRNA by Northern analysis and production of functional CK beta-11 by chemotaxis of human NK cells to C3L5-CK beta 11 supernatant. Only 10% of mice injected with C3L5-CK beta 11 developed tumors, compared with 100% of mice injected with a transduced control C3L5 line (C3L5-G1N). Importantly, the in vitro growth characteristics of the CK beta-11-transduced cell line were unaffected, suggesting the difference in growth in vivo was a result of chemokine production. Vaccination with C3L5-CK beta 11 partially protected animals from parental C3L5 challenge. Immunodepletion with anti-asialo-GM1 or anti-CD4 during C3L5-CK beta 11 vaccination significantly reduced CK beta-11 antitumor activity compared with control and anti-CD8-treated groups. Splenocytes from NK-depleted animals transferred the acquired immunity generated with C3L5-CK beta 11 vaccination, while splenocytes from the CD4-depleted animals did not. These results indicate, for the first time, that expression of CK beta-11 in a breast cancer cell line mediates rejection of the transduced tumor through a mechanism involving NK and CD4+ cells. Furthermore, CK beta-11-transduced tumor cells generate long-term antitumor immunity that requires CD4+ cells. These studies demonstrate the potential role of CK beta-11 as an adjuvant in stimulating antitumor responses.
The Journal of Immunology 05/2000; 164(8):4025-31. · 5.52 Impact Factor