Lilith Reeves

Stem Cell Collection and Gene Transfer in Fanconi Anemia

Fanconi anemia (FA) is a rare genetic syndrome characterized by progressive bone marrow failure (BMF), congenital anomalies, and a predisposition to malignancy. Successful gene transfer into hematopoietic stem cells (HSCs) could reverse BMF in this disease. We developed clinical trials to determine whether a sufficient number of CD34+ stem cells could be collected for gene modification and to evaluate the safety and efficacy of HSC-corrective gene transfer in FA genotype A (FANCA) patients. Here, we report that FA patients have significant depletion of their BM CD34+ cell compartment even before severe pancytopenia is present. However, oncoretroviral-mediated ex vivo gene transfer was efficient in clinical scale in FA-A cells, leading to reversal of the cellular phenotype in a significant percentage of CD34+ cells. Re-infusion of gene-corrected products in two patients was safe and well tolerated and accompanied by transient improvements in hemoglobin and platelet counts. Gene correction was transient, likely owing to the low dose of gene-corrected cells infused. Our early experience shows that stem cell collection is well tolerated in FA patients and suggests that collection be considered as early as possible in patients who are potential candidates for future gene transfer trials.

Safety Testing for Replication-Competent Retrovirus Associated with Gibbon Ape Leukemia Virus-Pseudotyped Retroviral Vectors

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.

Packaging cell line characteristics and optimizing retroviral vector titer: the National Gene Vector Laboratory experience.

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.

Defective homing is associated with altered Cdc42 activity in cells from patients with Fanconi anemia group A

Previous studies showed that Fanconi anemia (FA) murine stem cells have defective reconstitution after bone marrow (BM) transplantation. The mechanism underlying this defect is not known. Here, we report defective homing of FA patient BM progenitors transplanted into mouse models. Using cells from patients carrying mutations in FA complementation group A (FA-A), we show that when transplanted into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) recipient mice, FA-A BM cells exhibited impaired homing activity. FA-A cells also showed defects in both cell-cell and cell-matrix adhesion. Complementation of FA-A deficiency by reexpression of FANCA readily restored adhesion of FA-A cells. A significant decrease in the activity of the Rho GTPase Cdc42 was found associated with these defective functions in patient-derived cells, and expression of a constitutively active Cdc42 mutant was able to rescue the adhesion defect of FA-A cells. These results provide the first evidence that FA proteins influence human BM progenitor homing and adhesion via the small GTPase Cdc42-regulated signaling pathway.

A pilot study of dose-intensified procarbazine, CCNU, vincristine for poor prognosis brain tumors utilizing fibronectin-assisted, retroviral-mediated modification of CD34+ peripheral blood cells with O6-methylguanine DNA methyltransferase

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.

Detection of Ecotropic Replication-Competent Retroviruses: Comparison of S+/L- and Marker Rescue Assays

Guidelines for testing gene therapy products for ecotropic replication-competent retrovirus (Eco-RCR) have not been delineated as they have for amphotropic viruses. To evaluate biologic assays that can detect these viruses, we compared an S(+)/L(-) assay and a marker rescue assay designed specifically for Eco-RCR detection. Moloney murine leukemia virus (Mo-MuLV) obtained from the American Type Culture Collection was used as the positive control. For marker rescue, NIH 3T3 cells were transduced with a retroviral vector expressing the neomycin phosphotransferase gene (3T3/Neo). Inoculation and passage of test material in 3T3/Neo cells for 3 weeks (amplification) and subsequent testing in the S(+)/L(-) assay or the marker rescue assay increased the level of sensitivity for virus detection greater than 10-fold compared with direct inoculation of D56 S(+)/L(-) cells. When serial dilutions of Mo-MuLV stock were evaluated, six of six cultures had detectable virus by the S(+)/L(-) and marker rescue assays at dilutions of 10(-5) and 10(-6). At the 10(-7) dilution, five of six assays had detectable virus in both assays. The ability to detect virus-infected cells was also evaluated in a modification that substituted cells for supernatant. Fifteen 3T3/Neo cultures inoculated with 10(6) 293 cells containing 100 or 10 Mo-MuLV/3T3 cells were all positive by marker rescue. For dilution with 1 virus-infected cell per 10(6) 293 cells, 10 of 15 cultures were positive. At the 0.1-cell dilution only 2 of 15 cultures were positive. If we hope to detect one infected cell in a test article, the probability of detecting virus if the assay is performed in triplicate is 96.3%. In summary, after 3 weeks of amplification the S(+)/L(-) and marker rescue assays can detect virus with similar sensitivities. We prefer the marker rescue assay because of the more reliable growth features of NIH 3T3 cells compared with the D56 cell line. For laboratories analyzing clinical materials, this report may prove useful in establishing detection assays for Eco-RCR.

Production of retroviral vectors for clinical use.

Retroviral vectors were the first viral vectors to enter clinical trials and continue to be attractive candidates for applications where integration of the transgene is required. While these vectors are versatile and are used widely in the research setting, large-scale production for human use poses various challenges to insure quality and high titer. Our vector production facility has produced and certified over 20 vectors for clinical use and continues to be challenged to adapt the ever-changing vector technology to a method of production that complies with Good Manufacturing Practice (GMP). We describe two manufacturing methods for producing material for Phase I/II clinical trials and suggest ways for investigators to adapt these methods for multiple applications.

Copy Number Determination of Genetically-Modified Hematopoietic Stem Cells

Human gene transfer with gammaretroviral, murine leukemia virus (MLV) based vectors has been shown to effectively insert and express transgene sequences at a level of therapeutic benefit. However, there are numerous reports of disruption of the normal cellular processes caused by the viral insertion, even of replication deficient gammaretroviral vectors. Current gammaretroviral and lentiviral vectors do not control the site of insertion into the genome, hence, the possibility of disruption of the target cell genome. Risk related to viral insertions is linked to the number of insertions of the transgene into the cellular DNA, as has been demonstrated for replication competent and replication deficient retroviruses in experiments. At high number of insertions per cell, cell transformation due to vector induced activation of proto-oncogenes is more likely to occur, in particular since more than one transforming event is needed for oncogenesis. Thus, determination of the vector copy number in bulk transduced populations, individual colony forming units, and tissue from the recipient of the transduced cells is an increasingly important safety assay and has become a standard, though not straightforward assay, since the inception of quantitative PCR.

Vector Production in an Academic Environment: A Tool to Assess Production Costs

Generating gene and cell therapy products under good manufacturing practices is a complex process. When determining the cost of these products, researchers must consider the large number of supplies used for manufacturing and the personnel and facility costs to generate vector and maintain a cleanroom facility. To facilitate cost estimates, the Indiana University Vector Production Facility teamed with the Indiana University Kelley School of Business to develop a costing tool that, in turn, provides pricing. The tool is designed in Microsoft Excel and is customizable to meet the needs of other core facilities. It is available from the National Gene Vector Biorepository. The tool allows cost determinations using three different costing methods and was developed in an effort to meet the A21 circular requirements for U.S. core facilities performing work for federally funded projects. The costing tool analysis reveals that the cost of vector production does not have a linear relationship with batch size. For example, increasing the production from 9 to18 liters of a retroviral vector product increases total costs a modest 1.2-fold rather than doubling in total cost. The analysis discussed in this article will help core facilities and investigators plan a cost-effective strategy for gene and cell therapy production.

Partnership between CTSI and Business Schools Can Promote Best Practices for Core Facilities and Resources

Biomedical research enterprises require a large number of core facilities and resources to supply the infrastructure necessary for translational research. Maintaining the financial viability and promoting efficiency in an academic environment can be particularly challenging for medical schools and universities. The Indiana Clinical and Translational Sciences Institute sought to improve core and service programs through a partnership with the Indiana University Kelley School of Business. The program paired teams of Masters of Business Administration students with cores and programs that self‐identified the need for assistance in project management, financial management, marketing, or resource efficiency. The projects were developed by CTSI project managers and business school faculty using service‐learning principles to ensure learning for students who also received course credit for their participation. With three years of experience, the program demonstrates a successful partnership that improves clinical research infrastructure by promoting business best practices and providing a valued learning experience for business students.