Myeloproliferative Sarcoma Virus Research Articles

Long-term outcomes after gene therapy for adenosine deaminase severe combined immune deficiency

AbstractPatients lacking functional adenosine deaminase activity have severe combined immunodeficiency (ADA SCID), which can be treated with ADA enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (HSCT), or autologous HSCT with gene-corrected cells (gene therapy [GT]). A cohort of 10 ADA SCID patients, aged 3 months to 15 years, underwent GT in a phase 2 clinical trial between 2009 and 2012. Autologous bone marrow CD34+ cells were transduced ex vivo with the MND (myeloproliferative sarcoma virus, negative control region deleted, dl587rev primer binding site)–ADA gammaretroviral vector (gRV) and infused following busulfan reduced-intensity conditioning. These patients were monitored in a long-term follow-up protocol over 8 to 11 years. Nine of 10 patients have sufficient immune reconstitution to protect against serious infections and have not needed to resume ERT or proceed to secondary allogeneic HSCT. ERT was restarted 6 months after GT in the oldest patient who had no evidence of benefit from GT. Four of 9 evaluable patients with the highest gene marking and B-cell numbers remain off immunoglobulin replacement therapy and responded to vaccines. There were broad ranges of responses in normalization of ADA enzyme activity and adenine metabolites in blood cells and levels of cellular and humoral immune reconstitution. Outcomes were generally better in younger patients and those receiving higher doses of gene-marked CD34+ cells. No patient experienced a leukoproliferative event after GT, despite persisting prominent clones with vector integrations adjacent to proto-oncogenes. These long-term findings demonstrate enduring efficacy of GT for ADA SCID but also highlight risks of genotoxicity with gRVs. This trial was registered at www.clinicaltrials.gov as #NCT00794508.

Promoter usage regulating the surface density of CAR molecules may modulate the kinetics of CAR-T cells in vivo

Although chimeric antigen receptor T (CAR-T) cell therapy achieves high remission rates, challenges (e.g., toxicity management and relapse prevention) remain. The major risks are cytokine release syndrome and related neurological toxicity. The influence of the CAR surface density on the efficacy/safety of CAR-T cell therapy and the factors determining CAR density were not elucidated comprehensively. Here, we discovered that the use of the MND promoter increased the transduction rate and reduced the CAR surface density. Additionally, MND-driven CAR-T cells had prolonged antileukemia activity in a mouse model. In an initial dual-armed anti-CD19 CAR-T cell pilot study (ClinicalTrials.gov: NCT03840317), eight and six subjects were infused with MND and EF1α promoter-driven autologous CAR-T cells (3 × 105 CAR-T cells/kg), respectively. MND subjects developed mild fever and lower interferon gamma (IFN-γ) concentrations than in the EF1A19 group. All but one subject in each cohort reached minimal residual disease (MRD)-negative complete remission after the first month of evaluation. These results represent the first comprehensive study on the promoter-driven modulation of CAR-T cell functionality. These findings encourage further evaluation of the potential of the MND promoter to drive CAR-T cells as a broadly applicable cellular product for anticancer immunotherapy.

Surface Density of CAR Molecules May Modulate the Kinetics of CAR-T Cells In Vivo

Introduction: The various chimeric antigen receptor (CAR) genes used in most CAR-T clinical trials are expressed via the EF1α promoter. Recent reports have suggested that CAR expression using the myeloproliferative sarcoma virus enhancer, with the negative control region deleted and the dl587 Rev-binding site substituted (MND) promoter, to drive CAR expression results in a higher efficiency of gene transduction. The use of a different promoter for CAR expression is expected to alter the expression levels and the surface densities of CAR in transduced T cells. It has been reported previously that while only a few ligands are required to activate TCR signaling, CAR-T cells require approximately 100 CD19 molecules to trigger cytotoxic killing. Here we generated two CAR-T vectors (1904A and 1904B) using respectively the EF1α and the MND promoters to drive gene expression and compared them in cell culture and in a preclinical study. Methods: The second-generation CD19-CAR with T2A-linked tEGFR was expressed by using the EF1α or the MND promoter. The expression levels of tEGFR and CAR were determined by flow cytometry using the biotinylated-Erbitux, and the CD19-Fc protein and the α-FMC63scFv antibody, respectively. The in vitro cytotoxicity assay was performed by co-culture of CAR-T and Calcein-AM-stained K562-CD19. NOD-SCID mice engrafted with Raji-luciferase cells were used as an animal model to validate the activities of 1904A and 1904B CAR-T. A randomized, double-blinded, and dual-arms prospective clinical study was registered (NCT03840317) to compare the safety and efficacy of the treatments for recurrent/refractory (r/r) B-ALL. Subjects were assigned in two groups: those with blasts (by morphology) in bone marrow between 5-20% or above 20%. All subjects were preconditioned with fludarabine and cyclophosphamide before 3x105 CAR-T cells/kg infusion. Results: While tEGFR expression in 1904A and 1904B CAR-T cells was similar (with relative MFIs of 10.47±1.16 and 12.19±1.30 respectively), a significant increase in CAR expression was detected in 1904A compared to 1904B (with relative MFIs of 2.26±0.37 vs 1.24±0.07 and 4.37±0.59 vs 1.13±0.08 as determined by α-FMC63scFv and CD19Fc staining), indicating increased CAR expression on the surface of 1904A CAR-T cells. Importantly, 1904A cells showed greater cytotoxicity and release of cytokines, especially IFNγ and TNFα in cell culture. Both 1904A and 1904B treatments prolonged the median survival time in the animal model. Interestingly, the increase of tumor cells in mice was slower in the group treated with 1904B, suggesting 1904B T cells were more susceptible to the inhibitory effect of the tumor. In the clinical study, only 6 and 8 subjects for 1904A and 1904B respectively completed the trial by the end of July 2019. The median follow-up for 1904A and 1904B were 127 (67-168) and 138.5 (45-179) days, and tumor burdens were 3.06% (0.68%-82.7%) and 4.49% (1.29%-39.0%), respectively. The adverse events of subjects were monitored and recorded according to CTCAE 5.0. No significant difference in disease incidence or severity was detected between the two groups. The CR rate is 83.3% (5/6) for the 1904A group, and 100% (8/8) for the 1904B group. There were 3 (1904A) and 5 (1904B) subjects who bridged to HSCT. All subjects remained in remission with one CD19-negative recurrence in the 1904B group. Conclusion: Using the myeloproliferative sarcoma virus enhancer, we have generated a lentivirus vector, 1904B that can express CAR at a lower level on the cell surface, and investigated 1904B CAR-T functions in cell culture, in a mouse model, and in patients. Lower surface expression of CAR was found with reduced cytotoxicity and lower cytokine release of CAR-T in cell culture, but prolonged T cell activity in a mouse model. Human subjects treated with 1904B CAR-T developed fever later and shorter than 1904A, with slower expansion of CAR-T cells. We suggest that a lower surface expression of CAR molecules can decelerate the kinetics and decrease the intensity of CAR-T activation in vitro and in vivo. Figure Disclosures No relevant conflicts of interest to declare.

Retraction for Fusco et al., "A mos Oncogene-Containing Retrovirus, Myeloproliferative Sarcoma Virus, Transforms Rat Thyroid Epithelial Cells and Irreversibly Blocks Their Differentiation Pattern".

[This retracts the article DOI: 10.1128/JVI.56.1.284-292.1985.].

High Level of Perforin Expression Is Required for Effective Correction of Hemophagocytic Lymphohistiocytosis.

Perforin-1 mutations result in a potentially fatal hemophagocytic lymphohistiocytosis (HLH) with heightened immune activation, hypercytokinemia, pancytopenia, and end-organ damage. At present, hematopoietic stem cell (HSC) transplantation is curative, but limited by donor availability and associated mortality, making gene therapy an attractive alternative approach for HLH. We reported that perforin expression driven by cellular promoters in lentiviral (LV) vectors resulted in significant, albeit partial, correction of the inflammatory features in a murine model of HLH. We hypothesized that the level of perforin expression achieved per cell from ectopic moderate-strength cellular promoters (phosphoglycerate kinase gene/perforin-1 gene) is inadequate and thus engineered an LV vector using a viral promoter (MND; a modified Moloney murine leukemia virus long terminal repeat with myeloproliferative sarcoma virus enhancer) containing microRNA126 target sequences to restrict perforin expression in HSCs. We show here that the MND-LV vector restored perforin expression to normal levels in a perforin-deficient human natural killer cell line and perforin gene-corrected Perforin1-/- transplant recipients, whereas cellular promoters drove only partial correction. On lymphocytic choriomeningitis virus challenge, the clinical scores and survival improved only with the MND-LV vector, but inflammatory markers and cytotoxicity were improved with all LV vectors. Our studies suggest that although moderate levels of expression can result in partial amelioration of the HLH phenotype, high levels of perforin expression per cell are required for complete correction of HLH.

Optimized human CYP4B1 in combination with the alkylator prodrug 4-ipomeanol serves as a novel suicide gene system for adoptive T-cell therapies.

Engineering autologous or allogeneic T cells to express a suicide gene can control potential toxicity in adoptive T-cell therapies. We recently reported the development of a novel human suicide gene system that is based on an orphan human cytochrome P450 enzyme, CYP4B1, and the naturally occurring alkylator prodrug 4-ipomeanol. The goal of this study was to systematically develop a clinically applicable self-inactivating lentiviral vector for efficient co-expression of CYP4B1 as an ER-located protein with two distinct types of cell surface proteins, either MACS selection genes for donor lymphocyte infusions after allogeneic stem cell transplantation or chimeric antigen receptors for retargeting primary T cells. The U3 region of the myeloproliferative sarcoma virus in combination with the T2A site was found to drive high-level expression of our CYP4B1 mutant with truncated CD34 or CD271 as MACS suitable selection markers. This lentiviral vector backbone was also well suited for co-expression of CYP4B1 with a codon-optimized CD19 chimeric antigen receptor (CAR) construct. Finally, 4-ipomeanol efficiently induced apoptosis in primary T cells that co-express mutant CYP4B1 and the divergently located MACS selection and CAR genes. In conclusion, we here developed a clinically suited lentiviral vector that supports high-level co-expression of cell surface proteins with a potent novel human suicide gene.

Generation of Genetically Engineered Precursor T-Cells From Human Umbilical Cord Blood Using an Optimized Alpharetroviral Vector Platform.

Retroviral engineering of hematopoietic stem cell-derived precursor T-cells (preTs) opens the possibility of targeted T-cell transfer across human leukocyte antigen (HLA)-barriers. Alpharetroviral vectors exhibit a more neutral integration pattern thereby reducing the risk of insertional mutagenesis. Cord blood-derived CD34+ cells were transduced and differentiated into preTs in vitro. Two promoters, elongation-factor-1-short-form, and a myeloproliferative sarcoma virus variant in combination with two commonly used envelopes were comparatively assessed choosing enhanced green fluorescent protein or a third-generation chimeric antigen receptor (CAR) against CD123 as gene of interest. Furthermore, the inducible suicide gene iCaspase 9 has been validated. Combining the sarcoma virus-derived promoter with a modified feline endogenous retrovirus envelope glycoprotein yielded in superior transgene expression and transduction rates. Fresh and previously frozen CD34+ cells showed similar transduction and expansion rates. Transgene-positive cells did neither show proliferative impairment nor alteration in their lymphoid differentiation profile. The sarcoma virus-derived promoter only could express sufficient levels of iCaspase 9 to mediate dimerizer-induced apoptosis. Finally, the CD123 CAR was efficiently expressed in CD34+ cells and proved to be functional when expressed on differentiated T-cells. Therefore, the transduction of CD34+ cells with alpharetroviral vectors represents a feasible and potentially safer approach for stem cell-based immunotherapies for cancer.

166. Lentiviral Hematopoietic Stem Cell Gene Therapy for Sjögren-Larsson Syndrome

Sjogren-Larsson syndrome (SLS) is a rare autosomal recessive disorder characterized by scaling skin (ichthyosis), mental retardation, and spasticity. SLS is caused by mutations in the ALDH3A2 gene, which encodes fatty aldehyde dehydrogenase (FALDH), an enzyme that is involved in the oxidation of fatty aldehyde and fatty alcohol. In SLS, FALDH deficiency and impaired fatty aldehyde oxidation results in lipid accumulation, which is responsible for the symptoms. Spurred by previous hematopoietic stem cell gene therapy trials for multi-systemic diseases, such as Adrenoleukodystrophy (ALD), Metachromatic leukodystrophy (MLD), and Cystinosis, we investigated the plausibility of hematopoietic stem cell gene transfer to correct FALDH deficiency and alleviate the phenotype in SLS. We designed the hematopoietic stem cell gene therapy using a lentiviral vector containing human ALDH3A2 cDNA based on our previous MND-WASP (Wiskott-Aldrich syndrome protein) vector design. The lentiviral vector contains the normal human ALDH3A2 coding sequence which was derived from PCR products using human mobilized peripheral blood CD34+ cDNA. The vector was tested for FALDH protein expression in transfected human HEK293T cells by western blot and the enzyme activity was confirmed using HPLC-MS/UV. A transplantation experiment was performed using the Aldh3a2-/- KO mouse model, where the lineage negative BM cells from CD45.1 Aldh3a2-/- KO mice (C57BL/6 background) were sorted and transduced with a lentiviral vector followed by transplantation into lethally irradiated CD45.2 Aldh3a2-/- KO mice (C57BL/6 background). These mice are being monitored for cell engraftment, vector copy numbers, and any phenotypic changes. An initial analysis for the grafts showed 56% transduction in the MND(a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer)-GFP group and 31% transduction in the MND-ALDH3A2 group based on the CFU-C data. Flow cytometric analysis of peripheral blood on these mice at 6 and 12 post- transplant week showed 32% transduction in MND-GFP group. Further analysis including vector copy numbers and end point analysis for various tissues including liver, spleen, and brain are underway. This work provides the foundation for moving forward with potential lentiviral hematopoietic stem cell gene therapy for non-hematological disorders.

High Titer Lentivector from a Stable Lenti-Producer Efficiently Corrects CD34+ Hematopoietic Stem Cells from Patients with p47phox-Deficient Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) is an inherited immune deficiency due to mutations in the genes for the NADPH subunits (the genes for p47phox, p22phox, p67phox, p40phox autosomal chronic granulomatous disease), or gp91phox (X-linked chronic granulomatous disease). This results in a failure to generate phagocyte-derived superoxide and related reactive oxygen intermediates (ROIs), the major defect in chronic granulomatous disease causing recurrent infections and granulomatous complications. Hematopoietic stem cell transplantation (HSCT) with a suitable donor is potentially curative. However, in the absence of HLA-matched donor, gene therapy using autologous gene-corrected HSC offers potential for significant clinical benefit. To date, despite myeloid conditioning, gene therapy for CGD patients using gamma-retroviral vectors have achieved either minimal long-term gene marking and engraftment, or has been associated with insertional mutagenesis. In contrast, lentivector-mediated gene therapy has successfully treated patients with Wiskott-Aldrich syndrome and Metachromatic Leukodystrophy without any dysregulated clonal expansion.We used a lentivector construct which incorporates an MND internal promoter, a modified self-inactivating MoMuLV LTR U3 region with myeloproliferative sarcoma virus enhancer, and a 650bp single chicken b-globin insulator encoding codon-optimized p47phox gene. Mutations in p47phox accounts for the majority of AR-CGD. The production of large-scale, consistently-high-titer lentivector using a transient 4-plasmid transfection system however, is labor- and cost-prohibitive. To address this, we applied concatemeric array transfection of pCL20cW650 MND-p47-OPT into a stable packaging cell line (GPRTG) for HIV-based lentiviral vectors to create a stable producer of VSV-G pseudotyped pCL20cW650 MND-p47OP. The concatemer array of HIV lentiviral vector construct and bleomycin selectable gene cassette showed 10 copies of lentiviral vector in a stable producer line, capable of producing vector at 10^7 IU/ml.Hematopoietic CD34+ stem cells from p47phox- CGD were transduced with pCL20cW650 MND-p47-OPT vector (MOI 10) with 2 overnight transductions following 24 hours pre-activation with SCF, FLT-3L and TPO (100ng/ml). Following three weeks in vitro culture, non-transduced or transduced p47 CGD HSC versus normal HSC were 0%, 42% and 20% p47phox positive, respectively. To determine functional correction, PMA stimulated oxidant production was measured using the dihydrorhodamine assay, confirmation similar levels of oxidant generation in transduced patient cells compared with normal controls. More than 90% of CFU were vector positive, indicating a high level of gene marking. Transduced and control naïve p47phox-patient CD34+ HSC were transplanted into 20 immunodeficient Nodscid-gc deficient (NSG) mice, and at 13 weeks post-transplant the CD13+ human neutrophils arising in mouse bone marrow were assessed for p47phox expression. Over 40% CD13+ neutrophils expressed p47phox protein from NSG mice transplanted with transduced p47-patient CD34 HSC, compared with 74% or 0% in mice transplanted with normal CD34 or p47 patient naive CD34 cells respectively. Detailed histopathology of each transplanted mice confirmed the absence of vector insertion-related myeloid tumors, and deep sequencing of bone marrow CD45+ human cells from each mouse also demonstrated polyclonal distribution of vector integration sites. In conclusion, we provide preclinical data demonstrating the efficacy and safety of high titer VSVg-pseudotyped lentivector (CL20cW650 MND-p47-OPT) generated by our stable GPTRG p47 lenti-producer for correction of p47phox-deficient human CD34 HSC. DisclosuresNo relevant conflicts of interest to declare.

In Utero Lung Gene Transfer Using Adeno-Associated Viral and Lentiviral Vectors in Mice

Virus-mediated gene transfer to the fetal lung epithelium holds considerable promise for the therapeutic management of prenatally diagnosed, potentially life-threatening inherited lung diseases. In this study we hypothesized that efficient and life-long lung transduction can be achieved by in utero gene therapy, using viral vectors. To facilitate diffuse entry into the lung, viral vector was injected into the amniotic sac of C57BL/6 mice on embryonic day 16 (term, ∼ 20 days) in a volume of 10 μl. Vectors investigated included those based on adeno-associated virus (AAV) (serotypes 5, 6.2, 9, rh.64R1) and vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped HIV-1-based lentivirus (LV). All vectors expressed green fluorescent protein (GFP) under the transcriptional control of various promoters including chicken β-actin (CB) or cytomegalovirus (CMV) for AAV and CMV or MND (myeloproliferative sarcoma virus enhancer, negative control region deleted) for LV. Pulmonary GFP gene expression was detected by fluorescence stereoscopic microscopy and immunohistochemistry for up to 9 months after birth. At equivalent vector doses (mean, 12 × 10(10) genome copies per fetus) three AAV vectors resulted in long-term (up to 9 months) pulmonary epithelium transduction. AAV2/6.2 transduced predominantly cells of the conducting airway epithelium, although transduction decreased 2 months after vector delivery. AAV2/9-transduced cells of the alveolar epithelium with a type 1 pneumocyte phenotype for up to 6 months. Although minimal levels of GFP expression were observed with AAV2/5 up to 9 months, the transduced cells immunostained positive for F480 and were retrievable by bronchoalveolar lavage, confirming an alveolar macrophage phenotype. No GFP expression was observed in lung epithelial cells after AAV2/rh.64R1 and VSV-G-LV vector-mediated gene transfer. We conclude that these experiments demonstrate that prenatal lung gene transfer with AAV vectors engineered to target pulmonary epithelial cells may provide sustained long-term levels of transgene expression, supporting the therapeutic potential of prenatal gene transfer for the treatment of congenital lung diseases.

Anti-CD19 Chimeric Antigen Receptor Controlled By The Suicide Gene HSVsr39TK In Hematopoietic Stem Cells For Immunotherapy Of B-Lineage Malignancies

BackgroundAlthough significant improvements have been made, patients with relapsed or refractory B-cell malignancies continue to have unfavorable clinical outcomes. We hypothesize that transduction of hematopoietic stem cells (HSCs) with an anti-CD19 Chimeric Antigen Receptor (CAR) will produce a multi-lineage, persistent immunotherapy that can be controlled by the HSVsr39TK suicide gene. MethodsFirst generation anti-CD19 CAR lentiviral constructs containing the HSVsr39TK suicide gene were developed to compare vectors containing the human elongation factor alpha short (EFS) or myeloproliferative sarcoma virus U3 (MNDU3) promoters for transduction efficiency, antigen-specific cytotoxicity and ganciclovir (GCV)-induced cell death in primary human T-cells. The CD28 costimulatory domain was added to the selected construct, and high titer lentiviral vectors were generated to evaluate transduction of human umbilical cord blood (UCB) HSCs for in vitro and in vivo assays. In vitro assays were performed after culture under myeloid differentiation conditions, followed by assessment of phenotype, transduction efficiency, cytotoxic function and GCV-induced cell death. In vivo assays were conducted through transplantation of gene-modified human HSCs into irradiated NSG pups, compared to humanized NSG injected with non-modified human HSCs. Once engraftment was identified, mice from each cohort were further separated into GCV treated and untreated groups. Following GCV administration, mice were harvested to evaluate the presence of human and CAR-modified cells in the bone marrow, spleen and peripheral blood. ResultsIn human primary T cells, the MNDU3 promoter resulted in higher percentage of CAR expressing cells and mean fluorescence intensity compared to the EFS promoter. Cytotoxicity by the transduced T cells against the huCD19+Raji cell line showed similar target cell specific lysis among the constructs. Treatment with GCV effectively decreased the in vitro survival of the cells containing the HSVsr39TK gene compared to the non-transduced and control vector. The construct with MNDU3 promoter was then used with a CD28-containing second-generation anti-CD19 CAR (CCL-MND-αCD19/z/28-sr39). Once transduction efficiency and CAR function were validated in primary human T cells, this vector was used to transduce human UCB CD34+ cells. Following transduction, these cells were evaluated in vitro and in vivo. The cells used for the in vitro studies were cultured under myeloid differentiation conditions. The average number of CAR expressing cells was 45% at the clinically relevant vector copy number of 0.5-1 copies/cell. The myeloid cells transduced with the CCL-MND-αCD19/z/28-sr39 vector demonstrated CD19-specific killing and were eliminated by GCV. In vivo studies demonstrated successful engraftment of transduced HSC with CAR-expressing cells in the different hematopoietic lineages (T, NK, myeloid) detected among human cells in the bone marrow (1.2-15.4%, mean 7.6%), spleen (0.3-15.4%, mean 5.6%), and peripheral blood (0.5-30%, mean 9.2%). Mice engrafted with anti-CD19 CAR-modified HSCs exhibited decreased huCD19+ populations, compared to the mice engrafted with non-modified HSCs. Treatment with GCV resulted in significant decrease in CAR-expressing cells only in the mice transplanted with CD34+ cells transduced with the HSVsr39TK-containing vector. DiscussionHere we demonstrate that HSCs can be effectively transduced with an anti-CD19 CAR linked to the HSVsr39TK suicide gene. The CAR was detected in human cells in the bone marrow, spleen and peripheral blood and resulted in decreased B-lineage populations as an index of antigen-specific cytotoxicity; the HSVsr39TK gene conferred sensitivity to ganciclovir which eliminated transduced cells. These results provide pre-clinical support for the use of a CD19 targeted CAR in HSCs for the treatment of B-cell malignancies. Disclosures:Larson:Millenium: Speakers Bureau.

A retroviral vector suitable for ultrasound image-guided gene delivery to mouse brain

Gene transfer to the early-stage embryonic brain using the ultrasound image-guided gene delivery (UIGD) technique has proven to be valuable for investigating brain development. Thus far, this technology has been restricted to the study of embryonic neurogenesis. When this technique is designed to be employed for the study in adult animals, a long-term stable gene expression will be required. We attempted to develop a retroviral vector suitable for expressing exogenous genes in the brains of postnatal and adult mice in the context of the UIGD technique. Retroviral vectors containing four different long terminal repeats (LTRs) (each from Moloney murine leukemia virus (MoMLV), murine stem cell virus (MSCV), myeloproliferative sarcoma virus (MPSV) and spleen focus-forming virus (SFFV)) were compared using the well-known CE vector having the EF1α internal promoter as a control. The MS vector containing MSCV LTR produced a higher viral titer and a higher level of gene expression than other vectors including CE. The MS vector drove the gene expression in cultured neural stem cells for 3 weeks. Furthermore, the MS vector could efficiently deliver the gene to the mouse central nervous system, as transgene expression was found in various regions of the brains and spinal cords as well as in all major neural cell types. The data from an in vivo luciferase imaging analysis showed that the gene expression from the MS vector was sustainable for almost 3 months. Our data suggested that the MS vector would be suitable to construct mice containing the transgene expressed in the brain or spinal cord in a quick and cost-effective manner.

SIN Retroviral Vectors Expressing COL7A1 Under Human Promoters for Ex Vivo Gene Therapy of Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by loss-of-function mutations in COL7A1 encoding type VII collagen which forms key structures (anchoring fibrils) for dermal-epidermal adherence. Patients suffer since birth from skin blistering, and develop severe local and systemic complications resulting in poor prognosis. We lack a specific treatment for RDEB, but ex vivo gene transfer to epidermal stem cells shows a therapeutic potential. To minimize the risk of oncogenic events, we have developed new minimal self-inactivating (SIN) retroviral vectors in which the COL7A1 complementary DNA (cDNA) is under the control of the human elongation factor 1alpha (EF1alpha) or COL7A1 promoters. We show efficient ex vivo genetic correction of primary RDEB keratinocytes and fibroblasts without antibiotic selection, and use either of these genetically corrected cells to generate human skin equivalents (SEs) which were grafted onto immunodeficient mice. We achieved long-term expression of recombinant type VII collagen with restored dermal-epidermal adherence and anchoring fibril formation, demonstrating in vivo functional correction. In few cases, rearranged proviruses were detected, which were probably generated during the retrotranscription process. Despite this observation which should be taken under consideration for clinical application, this preclinical study paves the way for a therapy based on grafting the most severely affected skin areas of patients with fully autologous SEs genetically corrected using a SIN COL7A1 retroviral vector.

An Improved Bicistronic CD20/tCD34 Vector for Efficient Purification and In Vivo Depletion of Gene-Modified T Cells for Adoptive Immunotherapy

T-cell-based adoptive immunotherapy is widely used to treat graft rejection and relapse after stem cell transplantation (SCT). However, this approach is hampered by a high risk of life-threatening graft-versus-host-disease (GvHD). Clinical trials have demonstrated the value of suicide genes to modify T cells for the effective control of GvHD. Herewith, we show that the combination of a codon-optimized B-cell antigen (CD20op) with a selection marker based on a cytoplasmic truncated version of the human stem cell antigen CD34 (tCD34) allows the generation of highly enriched gene-modified T cells. We demonstrate coordinate co-expression of both transgenes and high expression of CD20op resulting in an increased susceptibility to Rituximab (RTX)-induced cell death. In addition, T cells partially retained their alloreactive potential and their CD4/CD8 ratio after transduction and expansion. Long-lasting transgene expression was sustained in vivo after adoptive transfer into Rag-1(-/-) mice. Moreover, gene-modified T cells were quickly and efficiently depleted from peripheral blood (PB) and secondary lymphoid organs of transplanted animals after RTX treatment. These results warrant further steps toward a clinical application of CD20op as a suicide gene for adoptive immunotherapy.

Optimal promoter usage for lentiviral vector-mediated transduction of cultured central nervous system cells

Lentiviral vectors transduce both dividing and non-dividing cells and can support sustained expression of transgenes. These properties make them attractive for the transduction of neurons and other neural cell types in vitro and in vivo. Lentiviral vectors can be targeted to specific cell types by using different promoters in the lentiviral shuttle vector. Even with identical constructs, however, levels of expression can vary significantly in different types of neurons and different culture preparations; expression levels in the same neuronal subtypes can be very different in primary cell culture and in vivo. We systematically assessed the ability of different promoters to direct expression of foreign transgenes in primary murine neocortical neurons, cerebellar granule cells and in undifferentiated and differentiated neuroblastoma cells. In primary cortical neurons, constructs using the ubiquitin C promoter directed the highest level of transgene expression; the phosphoglycerate kinase (PGK) promoter also directed robust transgene expression, while the cytomegalovirus (CMV) and MND (a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer) promoters resulted in the expression of the transgenes in only limited number of neurons. In contrast, in cerebellar granule cells and in differentiated SH-SY5Y neuroblastoma cultures, the CMV promoter directed the most robust transgene expression. There was similar variability in transgene expression directed by these promoters in primary cultures of oligodendrocytes and astrocytes. These findings may prove useful in the design of lentiviral vectors for use in cell culture models of the nervous system.

Stable Transgene Expression in Primitive Human CD34+Hematopoietic Stem/Progenitor Cells, Using theSleeping BeautyTransposon System

Sleeping Beauty (SB) transposon-mediated integration has been shown to achieve long-term transgene expression in a wide range of host cells. In this study, we improved the SB transposon-mediated gene transfer system for transduction of human CD34(+) stem/progenitor cells by two approaches: (1) to increase the transposition efficacy, a hyperactive mutant of SB, HSB, was used; (2) to improve the expression of the SB transposase and the transgene cassette carried by the transposon, different viral and cellular promoters were evaluated. SB components were delivered in trans into the target cells by Nucleoporation. The SB transposon-mediated integration efficacy was assessed by integrated transgene (enhanced green fluorescent protein [eGFP]) expression both in vitro and in vivo. In purified human cord blood CD34(+) cells, HSB achieved long-term transgene expression in nearly 7-fold more cells than the original SB transposase. Significantly brighter levels of eGFP expression (5-fold) were achieved with the human elongation factor 1alpha (EF1-alpha) promoter in Jurkat human T cells, compared with that achieved with the modified myeloproliferative sarcoma virus long terminal repeat enhancer-promoter (MNDU3); in contrast, the MNDU3 promoter expressed eGFP at the highest level in K-562 myeloid cells. In human CD34(+) cord blood cells studied under conditions directing myeloid differentiation, the highest transgene integration and expression were achieved using the EF1-alpha promoter to express the SB transposase combined with the MNDU3 promoter to express the eGFP reporter. Stable transgene expression was achieved at levels up to 27% for more than 4 weeks of culture after improved gene transfer to CD34(+) cells (average, 17%; n = 4). In vivo studies evaluating engraftment and differentiation of the SB-modified human CD34(+) cells demonstrated that SB-modified human CD34(+) cells engrafted in NOD/SCID/gamma chain(null) (NSG) mice and differentiated into multilineage cell types with eGFP expression. More importantly, secondary transplantation studies demonstrated that the integrated transgene was stably expressed in more primitive CD34(+) hematopoietic stem cells (HSCs) with long-term repopulating capability. This study demonstrates that an improved HSB gene transfer system can stably integrate genes into primitive human HSCs while maintaining the pluripotency of the stem cells, which shows promise for further advancement of non-virus-based gene therapy using hematopoietic stem cells.

Lentiviral-mediated correction of MPS VI cells and gene transfer to joint tissues

Joint disease in mucopolysaccharidosis type VI (MPS VI) remains difficult to treat despite the success of enzyme replacement therapy in treating other symptoms. In this study, the efficacy of a lentiviral vector to transduce joint tissues and express N-acetylgalactosamine-4-sulphatase (4S), the enzyme deficient in MPS VI, was evaluated in vitro and the expression of β-galactosidase was used to evaluate transduction in vivo. High viral copy number was achieved in MPS VI fibroblasts and 4-sulphatase activity reached 12 times the normal level. Storage of accumulated glycosaminoglycan was reduced in a dose dependent manner in both MPS VI skin fibroblasts and chondrocytes. Enzyme expression was maintained in skin fibroblasts for up to 41 days. Comparison of two promoters; the murine phosphoglycerate kinase gene promoter (pgk) and the myeloproliferative sarcoma virus long terminal repeat promoter (mpsv), demonstrated a higher level of marker gene expression driven by the mpsv promoter in both chondrocytes and synoviocytes in vitro. When injected into the rat knee, the expression of β-galactosidase from the mpsv promoter was widespread across the synovial membrane and the fascia covering the cruciate ligaments and meniscus. No transduction of chondrocytes or ligament cells was observed. Transduction was maintained for at least 8 weeks after injection. These results indicate that the lentiviral vector can be used to deliver 4S to a range of joint tissues in vitro and efficiently transduce synovial cells and express β-galactosidase in vivo.

Generating of Human CD4+ and CD8+ T Lymphocytes toward NY-ESO-1 by T Cell Receptor (TCR) Modification and Transduction for Adoptive TCR Gene-Transfer

The Cancer Testis (CT) antigen NY-ESO-1 is one of the most immunogenic cancer antigens eliciting strong humoral and cellular immune responses in tumor patients and therefore it is a promising candidate antigen for successful adoptive T cell transfer. The aim of our studies is the transfer of autologous T cells re-directed towards CT antigens by T cell receptor (TCR) gene transfer. The first precondition for genetic transfer of CT-Ag-specific TCRs is the availability of tumor-reactive CD4+ and CD8+ T cell clones that express a CT-Ag-specific TCR. Therefore, we generated the autologous CD8+ T cell clone ThP2 through stimulating HLA-A2.1− PBMCs with autologous HLA-A2+DCs loaded with synthetic NY-ESO-1157–165. After two restimulations, FACS-sorting and cloning, the T cell line specifically recognized the NY-ESO-1157–165 peptide and also specifically lysed NY-ESO-1157–165 expressing tumor cells. In addition, we generated NY-ESO-1 specific T helper1 clones from HLA-DR1+ and HLA-DR4+ healthy donors by stimulation of CD4+ T cells with autologous dendritic cells (DC) pulsed with the NY-ESO-187–111 peptide. The specificity of CD4+ T helper cell clones was determined by proliferation assays and IFN gamma ELISPOT through screening with the NY-ESO-187–111 peptide. By limiting dilution of the NYESO- 1-specific T cell populations we succeeded to isolate CD4+ T cell clones, which recognized NY-ESO-1-pulsed target cells and DCs pulsed with NY-ESO-1 protein. The second precondition for TCR gene transfer is the availability of efficient vector systems. Using vectors based upon mouse myelo-proliferative sarcoma virus (MPSV), it was possible to achieve a high transgene expression in the TCR-transduced T cells. Therefore, we cloned the TCR of the HL-A2-restricted NY-ESO-1-specific CTL clone ThP2 in the retroviral vector and documented the correct expression of the TCR-chains using peptide/HLA-multimers following retroviral transduction of peripheral PBMCs. Moreover, the NY-ESO-1 specific lysis of HLA-A2+ NY-ESO-1+ tumor cell lines after transduction in primary T cells was as well effective as the primary T cell clone. Because the expression of naive transgenic T cell receptors in recipient human T cells is often insufficient to achieve highly reactive T cell bulks we modified the TCR of the ThP2 CTL clone by, murinisation, codon optimalization or by introducing cysteins into the constant regions. Afterwards we compared the expression efficiency of the three different modifications on naive T cells by tetramer-staining. We were able to show that codon optimalization leads to an increase in the expression levels of the transgenic TCRs in human CD8+ T cells. The next step is the development of T cell transfer regiments, which are based on class-II-restricted TCR-transduced T cells.

Hematopoietic Stem Cell Gene Therapy Trial with Lentiviral Vector in X-Linked Adrenoleukodystrophy

We report preliminary results in 3 children with cerebral X-linked adrenoleukodystrophy (ALD) who received in September 2006, January 2007 and June 2008 lentiviral vector transduced autologous hematopoietic stem cell (HSC). We have previously demonstrated that cerebral demyelination associated with cerebral ALD can be stopped or reversed within 12–18 months by allogeneic HSC transplantation. The long term beneficial effects of HCT transplantation in ALD are due to the progressive turn-over of brain macrophages (microglia) derived from bone-marrow cells. For the current HSC gene therapy procedure, we used mobilized peripheral blood CD34+ cells that were transduced ex vivo for 18 hours with a non-replicative HIV1-derived lentiviral vector (CG1711 hALD) at MOI25 and expressing the ALD cDNA under the control of the MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substituted) promoter, and in the presence of 4 human recombinant cytokines (Il- 3, Stem Cell Factor [SCF], Flt3-ligand and Megakaryocyte Growth and Differentiation Factor [MGDF]) and CH-296 retronectine. Transduced cells were frozen to perform the required (RCL) safety tests. After thawing and prior to reinjection, 50%, 30% and 40% of transduced CD34+ cells expressed the ALD protein with a mean of 0.7, 0.6 and 0.65 copies of integrated provirus per cell. Transduced CD34+ cells were infused to ALD patients after a conditioning regimen including full doses of cyclophosphamide and busulfan. Hematopoietic recovery occured at day 13–15 post-transplant and the procedure was uneventful. In patient P1 and P2, the percentage of lymphocytes and monocytes expressing the ALD protein declined from day 60 to 6 months after gene therapy (GT) and remained stable up to 16 months post-GT. In P1, 9 to 13% of CD14+, CD3+, CD19+ and CD15+ cells expressed ALD protein 16 months post-transplant. In P2 and at the same time-point after transplant, 10 to 18% of CD14+, CD3+, CD19+ and CD15+ cells expressed ALD protein. ALD protein was expressed in 18–20% of bone marrow CD34+ cells from patients P1 and P2, 12 months post-transplant. In patient P3, 20 to 23% of CD3+, CD14+ and CD15+ cells expressed ALD protein 2 months after transplant. Tests assessing vector-derived RCL and vector mobilization were negative up to the last followups in the 3 patients. Integration of the vector was polyclonal and studies of integration sites arein progress. At 16 months post-transplant, HSC gene therapy resulted in neurological effects comparable with allogeneic HSC transplantation in patient P1 and P2. These results support that:ex-vivo HSC gene therapy using HIV1-derived lentiviral vector is not associated with the emergence of RCL and vector mobilization;a high percentage of hematopoietic progenitors were transduced expressing ALD protein in long term;no early evidence of selective advantage of the transduced ALD cells nor clonal expansion were observed.(This clinical trial is sponsored by Institut National de la Santé et de la Recherche Médicale and was conducted in part under a R&D collaboration with Cell Genesys, Inc., South San Francisco, CA)

Efficient Gene Marking with a Clinical Gammaretrovirus Vector Expressing MGMTP140K in Baboons and Macaques

Using gene therapy to protect hematopoietic stem cells (HSC) from alkylating agents used in the treatment of malignant disease is an attractive strategy to alleviate prolonged neutropenia and thrombocytopenia that is dose-limiting. Both adult and pediatric patients with glioblastomas urgently need improved therapeutic strategies since even with aggressive treatment the median survival after diagnosis is approximately 12 months. Chemotherapy with the nitrosourea BCNU, the methylating agents procarbazine or temozolomide, and other alkylating agents is effective and can prolong survival but the therapeutic benefit is attenuated due to hematopoietic toxicity which limits dose-escalation of these drugs. We have previously demonstrated in the dog and non-human primate model efficient gene marking, in vivo selection and chemoprotection from temozolomide and BCNU following transplantation with MGMTP140K gene-modified cells. Thus, we wanted to explore the efficacy of autologous transplantation in macaques and baboons using cells gene-modified with an MLV-based gammaretrovirus vector developed for a pending clinical trial. This retroviral vector contains a myeloproliferative sarcoma virus LTR, negative control region deleted, dl587rev primer binding site (MND) vector backbone, which expresses MGMTP140K from the 5′ LTR promoter, and is pseudotyped with the gibbon ape leukemia virus (GALV) envelope produced from Phoenix-GALV packaging cells (PhGALV-MND.GRS.P140K c38). Following transduction the gene marking in pre-infusion colony forming units (CFUs) was 74.3% and 69.8% in the macaque and baboon respectively as determined by CFU-PCR. Intracellular MGMT-staining of cultured baboon cells 4 (76.4%) and 11 (89.9%) days after transduction confirmed high gene transfer levels. Both animals recovered neutrophil and platelet levels within expected time frames relative to historical controls and the average provirus copy number determined by real-time PCR approximately one month after transplantation was 0.14 and 0.72 in the macaque and baboon respectively. Retrovirus integration site analysis in the macaque 90 days after transplantation, and before chemotherapy, confirmed polyclonal hematopoietic reconstitution. There was no indication of progression to a pre-leukemic state. The macaque has been treated twice with O6-benzylguanine (O6BG) and BCNU and the gene marking has stably increased approximately 2.5-fold. Aside from transient elevated liver enzymes following O6BG/BCNU treatment no additional extra-hematopoietic toxicity has been observed. In summary, we have been able to achieve efficient polyclonal gene marking with MGMTP140K gene-modified cells using a vector designed for clinical application in both macaques and baboons and have preliminary evidence of in vivo selection in the macaque. We believe that these large animal studies closely reflect a clinical setting and will help to further improve clinical HSC gene therapy.