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BACKGROUND: Severe combined immunodeficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) is a rare and life-threatening primary immunodeficiency. METHODS: We treated 50 patients with ADA-SCID (30 in the United States and 20 in the United Kingdom) with an investigational gene therapy composed of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with a self-inactivating lentiviral vector encoding human ADA. Data from the two U.S. studies (in which fresh and cryopreserved formulations were used) at 24 months of follow-up were analyzed alongside data from the U.K. study (in which a fresh formulation was used) at 36 months of follow-up. RESULTS: Overall survival was 100% in all studies up to 24 and 36 months. Event-free survival (in the absence of reinitiation of enzyme-replacement therapy or rescue allogeneic hematopoietic stem-cell transplantation) was 97% (U.S. studies) and 100% (U.K. study) at 12 months; 97% and 95%, respectively, at 24 months; and 95% (U.K. study) at 36 months. Engraftment of genetically modified HSPCs persisted in 29 of 30 patients in the U.S. studies and in 19 of 20 patients in the U.K. study. Patients had sustained metabolic detoxification and normalization of ADA activity levels. Immune reconstitution was robust, with 90% of the patients in the U.S. studies and 100% of those in the U.K. study discontinuing immunoglobulin-replacement therapy by 24 months and 36 months, respectively. No evidence of monoclonal expansion, leukoproliferative complications, or emergence of replication-competent lentivirus was noted, and no events of autoimmunity or graft-versus-host disease occurred. Most adverse events were of low grade. CONCLUSIONS: Treatment of ADA-SCID with ex vivo lentiviral HSPC gene therapy resulted in high overall and event-free survival with sustained ADA expression, metabolic correction, and functional immune reconstitution. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01852071, NCT02999984, and NCT01380990.)

Introduction: Access to cutting-edge technologies is essential for investigators to advance translational research. The Indiana Clinical and Translational Sciences Institute (CTSI) spans three major and preeminent universities, four large academic campuses across the state of Indiana, and is mandate to provide best practices to a whole state. Methods: To address the need to facilitate the availability of innovative technologies to its investigators, the Indiana CTSI implemented the Access Technology Program (ATP). The activities of the ATP, or any program of the Indiana CTSI, are challenged to connect technologies and investigators on the multiple Indiana CTSI campuses by the geographical distances between campuses (1–4 hr driving time). Results: Herein, we describe the initiatives developed by the ATP to increase the availability of state-of-the-art technologies to its investigators on all Indiana CTSI campuses, and the methods developed by the ATP to bridge the distance between campuses, technologies, and investigators for the advancement of clinical translational research. Conclusions: The methods and practices described in this publication may inform other approaches to enhance translational research, dissemination, and usage of innovative technologies by translational investigators, especially when distance or multi-campus cultural differences are factors to efficient application.

Background aims Gene therapy by autologous hematopoietic stem cell transplantation (HSCT) represents a new approach to treat sickle cell disease (SCD). Optimization of the manufacture, characterization and testing of the transduced hematopoietic stem cell final cell product (FCP), as well as an in depth in vivo toxicology study, are critical for advancing this approach to clinical trials. Methods Data are shown to evaluate and establish the feasibility of isolating, transducing with the Lenti/β AS3 -FB vector and cryopreserving CD34 + cells from human bone marrow (BM) at clinical scale. In vitro and in vivo characterization of the FCP was performed, showing that all the release criteria were successfully met. In vivo toxicology studies were conducted to evaluate potential toxicity of the Lenti/β AS3 -FB LV in the context of a murine BM transplant. Results Primary and secondary transplantation did not reveal any toxicity from the lentiviral vector. Additionally, vector integration site analysis of murine and human BM cells did not show any clonal skewing caused by insertion of the Lenti/β AS3 -FB vector in cells from primary and secondary transplanted mice. Conclusions We present here a complete protocol, thoroughly optimized to manufacture, characterize and establish safety of a FCP for gene therapy of SCD.

Lentiviral vectors have rapidly become a favorite tool for research and clinical gene transfer applications which seek to permanently introduce alterations in the genome. This status can be attributed primarily to their ability to transduce dividing as well as quiescent cells. When coupled with internal promotor selection to drive expression in one cell type but not another, the ease with which the vectors can be pseudotyped to either restrict or expand tropism offers unique opportunities previously unavailable to the researcher to manipulate the genome. Although LV can be produced from stable packaging cell lines and/or in suspension culture, by and far, most LV vectors are produced using adherent 293 T cells grown in plasticware and production plasmids transiently transfected with either PEI or Calcium Phosphate. The media is usually changed and un-concentrated vector supernatant collected between 24 and 48 h post-transfection. The supernatant may then be purified by Mustang Q chromatography, concentrated by Tangential Flow Filtration, and finally diafiltered into the final formulation buffer of choice. Here we describe a pilot scale method for the manufacture of a Lentiviral vector that purifies and concentrates approximately 6 L of un-concentrated LV supernatant to approximately 150 mL. Typical titers for most vector constructs range between 1 × 10

Lentiviral vectors have rapidly become a favorite tool for research and clinical gene transfer applications which seek to permanently introduce alterations in the genome. This status can be attributed primarily to their ability to transduce dividing as well as quiescent cells. When coupled with internal promotor selection to drive expression in one cell type but not another, the ease with which the vectors can be pseudotyped to either restrict or expand tropism offers unique opportunities previously unavailable to the researcher to manipulate the genome. Although LV can be produced from stable packaging cell lines and/or in suspension culture, by and far, most LV vectors are produced using adherent 293 T cells grown in plasticware and production plasmids transiently transfected with either PEI or Calcium Phosphate. The media is usually changed and un-concentrated vector supernatant collected between 24 and 48 h post-transfection. The supernatant may then be purified by Mustang Q chromatography, concentrated by Tangential Flow Filtration, and finally diafiltered into the final formulation buffer of choice. Here we describe a pilot scale method for the manufacture of a Lentiviral vector that purifies and concentrates approximately 6 L of un-concentrated LV supernatant to approximately 150 mL. Typical titers for most vector constructs range between 1 × 108 and 1 × 109 infectious particles per mL. This method may be performed reiteratively to increase total volume or can be further scaled up to increase yield.

Background: Severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID) is a rare disorder caused by ADA gene mutations, leading to lymphotoxic build-up of purine metabolites and profound immunodeficiency. Historically, enzyme replacement therapy (ERT) has been used as a bridge therapy until patients can receive an allogeneic hematopoietic stem cell transplantation (HSCT), ideally from a matched related donor (MRD) or, if none is identified, a non-matched and/or unrelated donor. We developed a self-inactivating lentiviral vector (LV), denoted EFS-ADA LV, encoding the human ADA cDNA sequence under the control of a shortened human elongation factor 1α gene promoter. A fresh or cryopreserved formulation of a drug product (OTL-101), composed of autologous hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with EFS-ADA LV, was evaluated in 2 prospective, non-randomized Phase I/II clinical trials at 2 USA centers. We report on safety and efficacy of OTL-101 in 30 ADA-SCID pediatric gene therapy (GT) subjects treated from 2013-2017 with a median follow up (FU) of 24 months (mo; range 12-26 mo), compared to a historical cohort of 26 ADA-SCID patients treated with HSCT. Methods: UCLA Fresh Study (NCT01852071): Autologous CD34+ HSPCs were isolated from bone marrow and pre-stimulated with cytokines before transduction with EFS-ADA LV to yield OTL-101, which was infused as a fresh formulation in 20 subjects (9 male, 11 female; aged 4 mo-4.3 yrs). Single dose busulfan (4 mg/kg) was administered prior to infusion of OTL-101. Subjects were followed for 24 mo. UCLA Cryo Study (NCT02999984): 10 subjects (4 male, 6 female; aged 5-15 mo) received a cryopreserved formulation of OTL-101, which allowed for an extended shelf-life and full quality control prior to infusion. Busulfan was administered in 2 doses, the first at 3 mg/kg and the second adjusted to target a total area under the curve of 4,900 µM*min (20 ng/mL*hr). At the time of analysis, all subjects reached 12 mo FU (except 1 subject who was withdrawn from the study due to lack of engraftment); 7 subjects reached 18 mo of FU. Historical Control Group: 26 patients (aged 0.2 mo-9.8 yrs) were treated with allogeneic HSCT (MRDs n=12, non-MRDs n=14) at Great Ormond Street Hospital, UK (n=16) or Duke University Children's Hospital, USA (n=10) from 2000-2016. Results: Sustained engraftment of genetically modified HSPCs was observed in 29/30 GT subjects by 6-8 mo and persisted through FU in both studies, based on vector gene marking in granulocytes and CD3+ T cell reconstitution (Figure). Subjects who engrafted maintained long-term metabolic detoxification from deoxyadenosine nucleotides after stopping ERT approximately 1 mo post-GT. At last FU (median 24 mo; range 12-24 mo) in the GT group, overall survival (OS) was 30/30 (100%) and event-free survival (survival in the absence of ERT reinstitution or rescue allogeneic HSCT; EvFS) was 29/30 (97%). OS and EvFS were higher in the GT group at last FU compared with HSCT controls (with or without an MRD) at 2 years (Table). One of 30 OTL-101 subjects (3%) did not engraft and was restarted on ERT; the subject was withdrawn from the study at 5.9 mo and subsequently received a rescue HSCT, whereas 42% of HSCT patients required rescue HSCT, PEG-ADA ERT or died. Among the 20 OTL-101 subjects in the UCLA Fresh Study who reached 2 years FU, 18 (90%) stopped immunoglobin replacement therapy (IgRT), compared to 52% of HSCT patients. Preliminary results were observed in 5/7 (71%) OTL-101 subjects in the UCLA Cryo Study with more limited (18 mo) FU. Twelve OTL-101 subjects experienced one or more serious adverse events, most frequently infections and gastrointestinal events; only 1 of which was considered treatment-related (bacteremia due to product contamination). In the GT group, there were no events of autoimmunity with ≤24 mo FU. Due to the autologous nature of OTL-101, there was no incidence of graft vs host disease (GvHD); in contrast, 8 HSCT patients experienced GvHD events (5 acute, 3 chronic events), 1 of which resulted in death. Conclusions: Based on sustained gene correction and restoration of immune function in all subjects who engrafted, treatment of ADA-SCID with OTL-101 has a favorable benefit-risk profile. Key correlates of engraftment were consistent across the expanded cohort. Importantly, higher rates of OS and EvFS compared with HSCT (with or without an MRD) were observed. Disclosures Kohn: Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Inventor on IP licensed from UC Regents to Orchard Therapeutics. Future royalties may occur., Research Funding; NIH: Research Funding. Shaw:Orchard Therapeutics: Consultancy, Other: Personal fees and non-financial support; NIH: Research Funding. Carbonaro-Sarracino:NIH: Other: Salary while working on project at UCLA 2013-2016, Research Funding; Orchard Therapeutics: Consultancy, Employment. De Oliveira:National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London: Research Funding; CIRM: Research Funding; National Gene Vector Repository: Research Funding; NIAID, NHI: Research Funding; Medical Research Council: Research Funding. Terrazas:California Institute for Regenerative Medicine: Research Funding; Gene Therapy Resource Program, NHLBI/NIH: Research Funding. Hollis:Curative Therapeutics: Consultancy, Other: Personal fees. Trevisan:Orchard Therapeutics: Research Funding. Arduini:Orchard Therapeutics: Employment, Equity Ownership. Lynn:Orchard Therapeutics: Employment, Equity Ownership. Kudari:Orchard Therapeutics: Employment, Equity Ownership. Spezzi:Orchard Therapeutics: Employment, Equity Ownership. Buckley:Duke University: Research Funding. Booth:SOBI: Consultancy; GSK: Honoraria; NovImmune: Consultancy. Thrasher:Generation Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; 4BIOCapital: Membership on an entity's Board of Directors or advisory committees; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Gaspar:Orchard Therapeutics: Employment, Equity Ownership, Patents & Royalties: Lentiviral vector for gene therapy of ADA-SCID.

Background: Genetic transfer of an anti-sickling β87-globin lentiviral vector (LV) into hematopoietic stem cells (HSC) followed by myeloablative transplant has cured one child with sickle cell anemia (SCA) (NEJM 2017), although it was not successful in 7 subsequent adult SCA patients, and modifications to intensify ablative conditioning, improve HSC dose, gene transfer are underway (Blood 130 Suppl 1: 527, 2017). Based upon our preclinical data (Blood 2009), we embarked upon a Reduced Intensity Conditioning (RIC) Phase I/II Pilot Study on Gene Transfer in Patients with SCA with a modified γ-Globin LV (NCT02186418), hypothesizing this approach will be safe, feasible and efficacious; Moreover, RIC will have significantly less toxicity, costs, and be implementable in many transplant centers, including those in some of the resource-poor countries, where supportive therapies for myeloablative transplants are unavailable, and where majority of SCA patients exist. Methods: Adult patients with severe SCA deemed eligible were transfused/erythrocytapheresed prior to HSC collection and transfused for 6 months post-transplant (PT) to Hb>10g/dl and HbS~30%. CD34+ HSC were collected via bone marrow harvest (BMH) and/or plerixafor mobilized Peripheral Blood Stem Collection (PBSC), selected for CD34+ cells and transduced. Patients received a single dose of IV melphalan (140mg/m2 BSA) 36hr prior to infusion of γ-globin modified (GM)-HSC. Patients were monitored for adverse events (AE), engraftment, vector copy number (VCN), modified HbF (HbF*) expression and clinical features of SCA. Results: Two SCA patients (35yo and 25yo) with HbS-β0 thalassemia genotype were treated. CD34+ HSC were collected via multiple BMH (P1) and BMH+PBSC (P2). Follow up data are available for 6 and 12mo on P1 and P2. P1 received 1x106 CD34+ cells/kgbw [vector copy number (VCN) 0.22], and P2 received 6.9x106 CD34+ cells/kgbw [VCN 0.46]. Time to neutrophil engraftment (ANC ≥ 500) was day 9 and 7 post-transplant (PT) in P1 and P2, respectively, and time to Plt recovery (Plt>50K) was day 14 PT in both. Patients included in this trial had severe disease and continued to have pre-existing chronic pain requiring significant opiates; hence ~80% of the AEs were pain events; other AEs were anticipated transient laboratory AEs associated with melphalan. Following GM-HSC infusion, both patients showed a progressive rise in HbF* (a point mutation in the γ-globin LV allows distinction from endogenous HbF by HPLC) starting from day 30 PT. Since patients had transfused HbA containing RBCs in the initial 6 months, HbF*/(HbF*+HbS) was calculated, and was 20% and 21% in P1 and P2 at day 180 PT and VCN 0.2-0.4, detected in all lineages. Integration site analysis, performed on the infused products (Day 0), at day 30 PT on P1 and P2, and on day 180 PT on P1 demonstrated highly polyclonal pattern of integration. At 1 yr PT, P1 had 20% HbF* (2.1g/dl HbF*, total Hb 10.6) with a stable VCN of 0.2-0.4 in multiple lineages in bone marrow and peripheral blood. The baseline Hb of P1 was 7.5-8.5g/dL prior to transplant. In the preceding 2 years prior to transplant, both patients were admitted for pain crises/acute chest >5-6 times/yr, and had chronic pain requiring chronic opiates. Chronic pain persisted for 4-5 months PT in P1, after which P1 has not required IV opiates, negligible oral opiates and has had no hospital visits/admissions with acute sickle events. P2 has required decreasing amounts of oral opiates for chronic back pain. Conclusions: Early results from 2 SCA adults treated with a modified γ-globin LV modified autologous HSC following RIC transplant showed excellent safety, feasibility, with minimal post-transplant toxicity, rapid count recovery, and sustained stable genetically modified cells in peripheral blood and bone marrow. The first patient shows significant clinical amelioration of the SCA phenotype at 1 year PT, with 20% vector-derived HbF (HbF*) that has caused amelioration of anemia, near elimination of chronic pain and absence of acute sickle events. The second patient, although still early post-transplant shows a similar HbF* trajectory. Additional study data will demonstrate whether this level of HbF* will provide consistent clinical benefit to patients with severe SCA. These early results, especially following a RIC transpant, are extremely promising; and if sustained, will provide a 'transportable' safe and feasible gene therapy for SCA. Disclosures Malik: CSL Behring: Patents & Royalties. Quinn:Global Blood Therapeutics: Research Funding; Silver Lake Research Corporation: Research Funding; Amgen: Research Funding.

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.

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.

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.

Although retroviral vectors are one of the most widely used vehicles for gene transfer, there is no uniformly accepted pre-clinical model defined to assess their safety, in particular their risk related to insertional mutagenesis. In the murine pre-clinical study presented here, 40 test and 10 control mice were transplanted with ex vivo manipulated bone marrow cells to assess the long-term effects of the transduction of hematopoietic cells with the retroviral vector MSCV-MGMT(P140K)wc. Test mice had significant gene marking 8-12 months post-transplantation with an average of 0.93 vector copies per cell and 41.5% of peripheral blood cells expressing the transgene MGMT(P140K), thus confirming persistent vector expression. Unexpectedly, six test mice developed malignant lymphoma. No vector was detected in the tumor cells of five animals with malignancies, indicating that the malignancies were not caused by insertional mutagenesis or MGMT(P140K) expression. Mice from a concurrent study with a different transgene also revealed additional cases of vector-negative lymphomas of host origin. We conclude that the background tumor formation in this mouse model complicates safety determination of retroviral vectors and propose an improved study design that we predict will increase the relevance and accuracy of interpretation of pre-clinical mouse studies.

Objective Lentiviral vectors are increasingly used for preclinical models of gene therapy and other forms of experimental transgenesis. Due to the broad tropism and the ability for concentration by ultracentrifugation, most lentiviral vector preparations are produced using the vesicular stomatitis virus glycoprotein (VSV-g) protein as envelope. Recently, Hanawa and colleagues have demonstrated that the ecotropic envelope protein of murine leukemia viruses allows efficient pseudotyping of HIV-1-derived vector particles. However, this method has found little acceptance, despite potential advantages. Materials and Methods We produced lentiviral vectors pseudotyped with murine ecotropic envelope using a four-plasmid transient transfection system and evaluated their performance in murine fibroblasts and hematopoietic cells. Results Titers of lentiviral "ecotropic" supernatants were only slightly lower than those produced with VSV-g, could be concentrated by overnight centrifugation (13,000 g ), and efficiently transduced murine fibroblasts and hematopoietic cells but not human cells. Our Institutional Biosafety Committee agreed on the production and use of replication-defective lentiviral vectors pseudotyped with murine ecotropic envelope under biosafety level 1 (BL1) conditions with additional BL2 practices. We also obtained useful guidelines for the work with human infectious lentiviral vectors. Conclusions For the researcher, "ecotropic" lentiviral vectors significantly improve the convenience of daily work, compared to the conditions required for lentiviral pseudotypes that are capable of infecting human cells. High efficiency and superior biosafety in combination with convenient handling will certainly boost the potential applicability of this important vector system.

Gene delivery using viral vectors is a powerful tool which can be used to correct genetic diseases and treat acquired illnesses. HIV-based lentiviral vectors (LVs) are advantageous in gene therapy applications for due to their genomic integration and ability to infect non-dividing cells. We have found that scale-up for is successful in retaining physical particle titers; however, the transduction efficiency of certain vectors, particularly in sensitive primary cells (e.g. human CD34+ cells), is significantly less than expected. Our working hypothesis is that current large-scale production methods may concentrate proteins that modulate transduction. To address this possibility, we characterized the “lentiviral vector proteome” of several different clinical vector lots using an Orbitrap Velos Pro Hybrid Mass Spectrophotometer. Since X-VIVO 10 medium was the final product medium in all LV lots, proteins identified in a sample of unused medium were eliminated from the analysis. We first identified a subset of 24 proteins common to ALL vector lots. Not surprisingly, further analysis showed that 6 of these proteins are both “top 25” exosome markers in the ExoCarta database and known gag-pol interacting proteins listed in the HIV-1 Human Interaction Database. Independent of production size and tangential flow filtration brand, we next identified 57 proteins unique to a low titer LV expressing human beta-globin (BG). We similarly identified 47 proteins unique to a high titer LV expressing adenosine deaminase (ADA). BG lots were enriched in proteasome subunit proteins and ADA lots in ribosomal subunit proteins. Interestingly, the ADA lots were also enriched in several 14-3-3 family proteins that are known to modulate intracellular signaling pathways. It is possible that the mere presence or relative levels of specific proteins, such as 14-3-3 proteins, can affect transduction. We can speculate that the mechanism involves indirect effects of the vector transgene on the producer cells which yields a particle that is more stable and/or has an increased capacity to transduce cells.

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 × 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.

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.

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.

Current regulatory guidelines require RCL testing of lentiviral vector preparations used in human clinical trials. Cells transduced ex vivo and cultured four days or more must also be tested before administering to the patient. Attenuated HIV (R8.71) can be considered as a theoretical RCL that could develop from a recombination event with wild-type HIV. Importantly, R8.71 lacks accessory proteins (vip, vif, vpr and nef) that are known to be essential for viral replication in T-lymphocytes. Based on this fact, the risk of a recombinant RCL being amplified in T-lymphocytes during an ex vivo transduction and expansion protocol is questionable. To test this possibility, we transduced human T-lymphocytes with one of two RCL-negative lentiviral vector preparations (eGFP or a chimeric antigen receptor (CAR)) that were spiked or not with R8.71 or wild-type HIV. C8166 cells served as a positive control for cells permissive to R8.71. The transduction and subsequent expansion lasted 10 days total and the experiment was performed on two independent occasions. Using day 10 supernatants from all groups, a full RCL assay (with amplification and indicator phases) was performed. Product enhanced reverse transcriptase (PERT) and P24 ELISA assays served as readouts for the RCL assay. Transduction rates were determined by flow cytometry and found to be 57-60% and 10-37% for the eGFP and CAR vector, respectively. The RCL assay results showed that all T-lymphocyte groups exposed to R8.71 were negative but those exposed to wild-type HIV were positive. C8166 cells exposed to R8.71 or wild-type HIV were positive in the RCL assay. These results suggest that a recombinant RCL that possess the wild-type HIV envelope would NOT amplify in human T-lymphocytes during a model ex vivo transduction procedure. The amplification of an arguably more dangerous VSVG-enveloped recombinant RCL is still possible; however, it could be reliably detected by quicker and less expensive assays such as qPCR to VSVG sequences.

Patients with X-linked severe combined immunodeficiency (SCID-X1) were successfully cured following gene therapy with a gamma-retroviral vector (gRV) expressing the common gamma chain of the interleukin-2 receptor (IL2RG). However, 5 of 20 patients developed leukemia from activation of cellular proto-oncogenes by viral enhancers in the long-terminal repeats (LTR) of the integrated vector. These events prompted the design of a gRV vector with self-inactivating (SIN) LTRs to enhance vector safety. Herein we report on the production of a clinical-grade SIN IL2RG gRV pseudotyped with the Gibbon Ape Leukemia Virus envelope for a new gene therapy trial for SCID-X1, and highlight variables that were found to be critical for transfection-based large-scale SIN gRV production. Successful clinical production required careful selection of culture medium without pre-added glutamine, reduced exposure of packaging cells to cell-dissociation enzyme, and presence of cations in wash buffer. The clinical vector was high titer; transduced 68-70% normal human CD34(+) cells, as determined by colony-forming unit assays and by xenotransplantation in immunodeficient NOD.CB17-Prkdc(scid)/J (nonobese diabetic/severe combined immunodeficiency (NOD/SCID)) and NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ (NOD/SCID gamma (NSG))) mice; and resulted in the production of T cells in vitro from human SCID-X1 CD34(+) cells. The vector was certified and released for the treatment of SCID-X1 in a multi-center international phase I/II trial.

Hemophilia A is a monogenic bleeding disorder currently treated by lifelong infusions of factor VIII (fVIII). The development of gene-based treatments for hemophilia A has been hampered by several biological hurdles including the low level expression of fVIII from genetically engineered target cells, the large transgene size, and transgene product immunogenicity. We now show that these major hurdles are overcome by i) using a bioengineered FVIII transgene (ET3) that expresses at 10 - 100-fold higher levels compared to other human FVIII sequences, ii) targeting hematopoietic stem cells (HSC) ex vivo using a self-inactivating (SIN) lentiviral vector (LV) encoding ET3, and iii) using a non-myeloablative conditioning regimen prior to HSC transplantation that results in transgene product associated immune tolerance. The clinical trial design and preclinical data were favorably reviewed by the RAC, Institutional Biosafety Committee, and FDA. However upon full-scale clinical production of the SIN-ET3-LV, an additional hurdle was discovered that resulted in low LV manufacturing titer, which we showed is due to promoter competition between the CMV/HIV-LTR and the internal promoter driving ET3 production (eg. EF1a). We show that high-titer recombinant LV encoding ET-3 can be achieved by replacing the EF1a internal promoter with one that has low activity in the 293T producer cells. CD68 is a monocyte-restricted promoter that is not active in 293T producer cell lines and thus minimizes promoter interference. Using a SIN-CD68-ET3-LV expression vector, high titer virus was generated using a laboratory scale production strategy as well as a 6 L scalable clinical LV production platform. Our scalable manufacturing process generated titers >2 x 109/ml, which is sufficient to support an ex vivo HSC transduction/transplantation pilot/phase I clinical trial. Late stage preclinical testing using clinically processed vector showed that the high titer virus efficiently transduces hematopoietic cell lines, as determined by quantitative PCR, and Southern analysis, which confirmed integrated transgene stability over 8 weeks. Efficient and stable gene transfer into murine Sca-1+ cells harvested from hemophilia A mice was also achieved. A dose response of LV addition to Sca-1 cells and FVIII expression was observed in transplanted mice. Expression of >40% normal FVIII levels was achieved at multiplicity of infections (MOIs) as low 20, and long term FVIII expression was observed post HSCT. No difference in the engraftment of genetically modified cells as compared to mock-modified cells was observed. In addition, efficient, stable and dose responsive gene marking (up to 0.6 VCN/cell) of human mobilized peripheral blood(mPB)-derived CD34+ cells was reproducibly achieved. Transduction of mPB-CD34+ cells at MOIs ranging from 1 to 125 had no effect on methylcellulose colony forming unit growth when comparing SIN-CD68-ET3-LV to non-modified cells. In addition, there was no effect on mPB-CD34+ cell engraftment of NSG mice when comparing modified and mock modified cells. Furthermore, next generation sequencing-based insertion site analysis showed integration of SIN-CD68-ET3-LV to be similar, with respect to integration events near oncogenes, to that of other vectors in current clinical trials. Collectively, this comprehensive, preclinical data set provides support for regulatory submissions to conduct a pilot/phase I clinical trial of a SIN-CD68-ET3-LV transduced-HSC transplantation product for the treatment of hemophilia A. Disclosures Doering: Bayer Healthcare: Consultancy, Honoraria, Research Funding; Expression Therapeutics: Equity Ownership. Denning:Expression Therapeutics: Employment. Spencer:Expression Therapeutics: Equity Ownership.

The need for γ-retroviral (gRV) vectors with a self-inactivating (SIN) design for clinical application has prompted a shift in methodology of vector manufacturing from the traditional use of stable producer lines to transient transfection-based techniques. Herein, we set out to define and optimize a scalable manufacturing process for the production of gRV vectors using transfection in a closed-system bioreactor in compliance with current good manufacturing practices (cGMP). The process was based on transient transfection of 293T cells on Fibra-Cel disks in the Wave Bioreactor. Cells were harvested from tissue culture flasks and transferred to the bioreactor containing Fibra-Cel in the presence of vector plasmid, packaging plasmids and calcium-phosphate in Dulbecco's modified Eagle's medium and 10% fetal bovine serum. Virus supernatant was harvested at 10-14 h intervals. Using optimized procedures, a total of five ecotropic cGMP-grade gRV vectors were produced (9 liters each) with titers up to 3.6 × 10(7) infectious units per milliliter on 3T3 cells. One GMP preparation of vector-like particles was also produced. These results describe an optimized process for the generation of SIN viral vectors by transfection using a disposable platform that allows for the generation of clinical-grade viral vectors without the need for cleaning validation in a cost-effective manner.

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.

Murine safety studies are routinely used for gathering preclinical safety and efficacy data and, for Phase I studies, Good Laboratory Practice (GLP) compliance is not mandated. However, extensive amounts of data must be gathered and analyzed. An inter-relational database is an effective tool for storing, sorting, and reviewing data.

This chapter will review the design and execution of release testing requirements for retroviral vectors and gene-modified cells consistent with ensuring the success of the clinical trial on the basis of current US regulatory requirements. It is the ethical and legal responsibility of the clinical trial sponsor(s) to ensure safety of the patients through proper evaluation of the drug products prior to use. Any clinical trial drug product used in human subjects must be produced and evaluated for safety, quality, purity, and effectiveness according to Current Good Manufacturing Practices appropriate for the stage of clinical development.

Murine safety studies are routinely used for gathering preclinical safety and efficacy data and, for Phase I studies, Good Laboratory Practice (GLP) compliance is not mandated. However, extensive amounts of data must be gathered and analyzed. An inter-relational database is an effective tool for storing, sorting, and reviewing data.

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.

This chapter will review the design and execution of release testing requirements for retroviral vectors and gene-modified cells consistent with ensuring the success of the clinical trial on the basis of current US regulatory requirements. It is the ethical and legal responsibility of the clinical trial sponsor(s) to ensure safety of the patients through proper evaluation of the drug products prior to use. Any clinical trial drug product used in human subjects must be produced and evaluated for safety, quality, purity, and effectiveness according to Current Good Manufacturing Practices appropriate for the stage of clinical development.

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.

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.

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.

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.

Fanconi anemia (FA) is a genetic syndrome characterized by the uniform development of aplastic anemia. Current therapy for patients lacking HLA-identical sibling hematopoietic stem cell (HSC) donors are not optimal. We have opened two phase I clinical trials to determine if potentially useful numbers of CD34+ cells can be collected early in the course of the disease (collection study) and if these cells, once corrected, can engraft without cytoreduction and demonstrate proliferative advantage in vivo over un-corrected cells (gene transfer study). To date, 7 FA patients have undergone a 20 ml/kg bone marrow harvest (BMH) with an average of 1.9|[times]|106 CD34+ cells/kg (range 0.3|[ndash]|2.9|[times]|106 CD34+ cells/kg) collected, suggesting that collection of adequate numbers of cells is possible but challenging, even early in the disease. In the gene transfer study, 3 FA patients with genotype A (FAA) have enrolled, meeting eligibility criteria of FAA, no evidence of malignancy and a minimum of 1|[times]|105 viable CD34+ cells/kg for ex vivo culture and gene transfer. BMHs from the 3 patients (2 fresh and one previously cryopreserved) were CD34+ cell selected. Despite collection before significant pancytopenia, an average of only 5|[times]|105 CD34+ cells/kg (range 1.5|[ndash]|10|[times]|105 CD34+ cells/kg) was purified from these 3 cases representing |[sim]|10% of the expected yield from normal individuals. These cells underwent ex vivo gene transfer using cytokine prestimulation in serum-free medium followed 2 exposures to a GALV-MSCV-FANCA vector. Transduction efficiency of the final product determined by real-time PCR analysis of progenitors averaged 48% (range 40|[ndash]|62%). Equivalent correction of mitomycin C hypersensitivity in progenitor cells confirmed this analysis at the functional level. Nucleated cell recovery after ex vivo manipulation was 82|[ndash]|110% of input nucleated cells using freshly harvested bone marrow derived CD34+ cells (N=2). However, despite good CD34+ cell recovery and viability after CD34+ selection, only 6% of input nucleated cells were recovered utilizing CD34+ cells purified from the previously cryopreserved bone marrow and these cells were not re-infused. In the two patients who did receive gene corrected cells, the total cell dose re-infused was 2.5|[ndash]|3.5|[times]|105 nucleated cells/kg, reflecting the low number of initial CD34+ cells placed in culture. FAA vector sequences were detected in the PB of one patient early post-infusion (+4 weeks) but none was detected after 8 weeks. Despite no long term detection of the corrective gene in the PB and BM, both patients exhibited a transient but marked improvement (10|[ndash]|20% increase) in their peripheral blood hemoglobin (> 6 months) and platelets counts (2|[ndash]|3 months). The data demonstrates that gene transfer efficiency in the clinical setting has been significantly improved, however the collection of adequate numbers of HSC may be the critical barrier to the success of genetic correction attempts in FA.

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 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.

Retroviral gene therapy vectors expressing the MGMTP140K transgene have been shown to protect hematopoietic cells from toxicity associated with a combined cancer treatment using 6-benzylguanine (6-BG) and an alkylating agent such as Temozolomide (TEM). In a Phase I gene transfer trial, high grade astrocytoma patients having poor prognoses using standard therapies, will undergo escalating dose treatments with 6-BG and TEM following MGMTP140K gene transfer into autologous hematopoietic stem cells. Although retroviral vectors are one of the most widely used vehicles for gene transfer, there is no uniformly accepted preclinical model defined to assess their safety, and, in particular their risk related to insertional mutagenesis. This study was designed as a murine pre-clinical study to assess the long term effects of the transduction of hematopoietic cells with the retroviral vector to be used in the clinical trial, MSCV-MGMTP140Kwc.

Fanconi anemia (FA) is a devastating blood disease associated with bone marrow (BM) failure. Currently the effective treatment for FA is BM transplantation. We and others showed that FA murine hematopoietic stem and progenitor cells (HSC/Ps) have defective hematopoietic reconstitution after BM transplantation. In addition, several clinical gene therapy trials in FA patients have failed to show sustained engraftment of FA HSC/Ps complemented with a functional FA gene. The mechanism underlying this engraftment defect remains unknown. To determine whether the poor engraftment of FA HSC/Ps observed in mouse knockout models and clinical gene therapy trials in FA patients results from an intrinsic defect of the FA HSC/Ps or an impaired microenvironment in the FA bone marrow, we have assessed the self-renewal ability of HSC/Ps and hematopoietic supportive capacity of BM stromal cells from FA-A patients. First, we performed quantitative cobblestone area-forming cell (CAFC) assays using MS-5 stromal layer. CAFCs were enumerated at 2 and 5 weeks of cocultures between BM mononuclear cells from a normal donor and three FA-A patients. Early-appearing CAFCs (week 2) represent transient repopulating cells equivalent to progenitor colony-forming units (CFUs), whereas late-appearing CAFCs (week 5) are representative of long-term repopulating stem cells. The frequency of FA-A CAFCs was 15–20 folds decreased compared with that of the normal donor at both week 2 and week 5. Second, we carried out long-term culture-initiating cell (LTC-IC) assays using BM mononuclear cells and BM-derived stromals from normal donors or FA-A patients. Compared to coculture with normal stromal cells, the recovery of colony forming cells (CFCs) that grew on FA-A stromal layer after 5–6 weeks incubation was significantly decreased for either normal or FA-A HSC/Ps (41.4% ± 3.1 CFC in normal HSC/Ps - normal stroma compared to 14.3% ± 1.6 CFC in normal HSC/Ps – FA-A stroma and 2.4 ± 0.6 CFC in FA-A HSC/Ps- FA-A stroma). Finally, we conducted CFC and cell-cell adhesion assays using BM-derived stromal cells from normal donors and FA-A patients. We observed that FA-A BM stroma failed to support HSC/P cells from either normal donors or FA-A patients. Specifically, severe adhesion defect was found in adhesion of normal and FA-A HSC/Ps with FA-A stromal layer (5.5% ± 0.6 CFC adhesion in normal HSC/Ps - normal stroma compared to 2.4% ± 0.5 CFC adhesion in normal HSC/Ps – FA-A stroma and 1.0 ± 0.7 CFC adhesion in FA-A HSC/Ps- FA-A stroma). These results suggest that both a cell-autonomous defect of FA HSC/Ps and an impaired FA BM microenvironment may contribute to the abnormal hematopoiesis in FA.

A significant percentage of clinical grade safety-enhanced gamma-retrovirus and lentivirus vector supernatants are currently produced by transient transfection. These products contain high concentrations of plasmid DNA that pose the risk of transfer into subjects, and preclude accurate estimation of transduction efficiency. Most envelope pseudotyped vectors are too labile for DNAse treatment or other purification steps and adding non-clinical grade reagents into any production step further complicates product qualification. Here we present development of a method to remove residual plasmid as a part of the transduction procedure of human CD34+ cells or mouse bone marrow progenitors, using the DNAse Pulmozyme®. Using a standard clinical transduction protocol, CD34+ were transduced with SRS11.EFS.IL2RG.pre* or SRS11.EFS.EGFP. pre* gamma-retroviral vectors pseudotyped with GALV envelope or a mock control. Retronectin coated flasks preloaded with vector were either treated with Pulmozyme® or no Pulmozyme®. Cells were transduced on two consecutive days and collected 4 –24 hrs after the 2nd transduction (day1). CFU were plated and enumerated and proportion of transduced progenitors quantified by real time PCR (qPCR) using wPRE primers. For the EGFP containing vector, gene transfer was determined by flow cytometry in cultures that were continued for up to 14 days. DNA was extracted and tested by qPCR on days 1, 7, and 14. Residual plasmid DNA was quantified using GALV primers. Transduction was also estimated on the bulk cultures using qPCR. In all qPCR reactions human ApoB was quantified concurrently to asses the cell number. Pulmozyme® treatment reduced GALV copy numbers in cells one day following transduction by up to 20-fold. After 7 days in culture, there was no residual plasmid detectable in all groups. More importantly, Pulmozyme® treatment did not alter the transduction efficiency of the progenitors in any of the experiments, as evaluated by either wPRE qPCR on Day-7 DNA (4.3 vs. 4.2, 6.8 vs. 7.6 and 2.0 vs. 1.9 vector copies per cell for Pulmozyme® treated and non-treated respectively), colony PCR (69.4 vs. 66.7% vector positive CFUs) or flow cytometry (81.8 vs. 85.7% EGFP positive cells). We also analyzed the engraftment potential of cells transduced using Pulmozyme®-treated versus non-treated vector after transplanting cells in NOD/SCID and NOD/SCID-IL2Rg null (NOG) mice. Analysis of mice at 6 weeks post transplantation showed no significant reduction in human cell engraftment in the Pulmozyme® and the average copy number per human cell in NOG bone marrow was 0.65 vs. 0.52 and in NOD/SCID bone marrow 1.03 vs. 0.67 (Pulmozyme® treated versus non-treated, respectively). We also tested Pulmozyme® treatment in mouse experiments using gamma-retroviral vector pseudotyped with ecotropoc envelope with a similar result. We conclude that treatment of gamma-retroviral vector supernatant with Pulmozyme® after vector preload on Retronectin® coated plates reduces vector and packaging plasmids and does not inhibit transduction, clonogenic potential, or engraftment. The removal of excess plasmid from transiently produced virus supernatant without affecting the virus transducibility allows accurate estimation of transduction efficiencies prior to transplant and will conceivably reduce the toxicity to primary cells from excess plasmid. These studies can be directly translated to preclinical animal safety studies and clinical trials.