Replication-competent Retrovirus Research Articles

Wnt Signaling Drives Correlated Changes in Facial Morphology and Brain Shape.

Canonical Wnt signaling plays multiple roles critical to normal craniofacial development while its dysregulation is known to be involved in structural birth defects of the face. However, when and how Wnt signaling influences phenotypic variation, including those associated with disease, remains unclear. One potential mechanism is via Wnt signaling’s role in the patterning of an early facial signaling center, the frontonasal ectodermal zone (FEZ), and its subsequent regulation of early facial morphogenesis. For example, Wnt signaling may directly alter the shape and/or magnitude of expression of the sonic hedgehog (SHH) domain in the FEZ. To test this idea, we used a replication-competent avian sarcoma retrovirus (RCAS) encoding Wnt3a to modulate its expression in the facial mesenchyme. We then quantified and compared ontogenetic changes in treated to untreated embryos in the three-dimensional (3D) shape of both the SHH expression domain of the FEZ, and the morphology of the facial primordia and brain using iodine-contrast microcomputed tomography imaging and 3D geometric morphometrics (3DGM). We found that increased Wnt3a expression in early stages of head development produces correlated variation in shape between both structural and signaling levels of analysis. In addition, altered Wnt3a activation disrupted the integration between the forebrain and other neural tube derivatives. These results show that activation of Wnt signaling influences facial shape through its impact on the forebrain and SHH expression in the FEZ, and highlights the close relationship between morphogenesis of the forebrain and midface.

Single Amino Acids G196 and R198 in hr1 of Subgroup K Avian Leukosis Virus Glycoprotein Are Critical for Tva Receptor Binding.

Avian leukosis viruses (ALVs), a type of retrovirus responsible for various tumor diseases in chickens, are divided into 11 subgroups: ALV-A to ALV-K. After the envelope glycoproteins of ALV interact with the cellular receptor to initiate viral invasion, alterations in a few amino acids of the viral glycoproteins or cell receptors may trigger changes in their conformation and binding affinity. To identify the functional determinants of the ALV-K envelope protein that binds to Tva (a recently identified cellular receptor of ALV-K), using the strategy of continuous, segment-by-segment substitution of the gp85-encoded surface glycoprotein (SU) of ALV-K GDFX0602 with ALV-E ev-1 (using Tvb as the receptor), a series of chimeric soluble gp85 proteins were expressed for co-immunoprecipitation (co-IP) analysis and a series of recombinant viruses with replication-competent avian retrovirus vectors containing Bryan polymerase (RCASBP) as their skeleton were created for transfecting to DF-1 cells and titer determination. The co-IP analysis, fluorescence-activated cell sorting, and virus titer measurements revealed that the substitution of residues 194–198, 206–216 of hr1, residues 251–256 between hr1 and hr2, and residues 269–280 of hr2 were identified to reduce the binding of gp85 to Tva. The substitution of residues 194–221 in hr1 nullified the infectiveness of these viruses, similar to the effect of single amino acid mutations in K251E and L252I located between hr1 and hr2; continuous amino acid mutations in hr2 could not produce the same effect despite reducing their infectiveness. Finally, single amino acid mutations G196A and R198H nearly abolished the binding of gp85 to Tva and nullified the infectiveness of these viruses to DF-1. This study paves the way for exploring the molecular mechanisms of the binding of Tva to ALV-K SU.

One-Year Follow-up of ZUMA-2, the Multicenter, Registrational Study of KTE-X19 in Patients with Relapsed/Refractory Mantle Cell Lymphoma

One-Year Follow-up of ZUMA-2, the Multicenter, Registrational Study of KTE-X19 in Patients with Relapsed/Refractory Mantle Cell Lymphoma

Outcomes of Patients (Pts) in ZUMA-9, a Multicenter, Open-Label Study of Axicabtagene Ciloleucel (Axi-Cel) in Relapsed/Refractory Large B Cell Lymphoma (R/R LBCL) for Expanded Access and Commercial Out-of-Specification (OOS) Product

Outcomes of Patients (Pts) in ZUMA-9, a Multicenter, Open-Label Study of Axicabtagene Ciloleucel (Axi-Cel) in Relapsed/Refractory Large B Cell Lymphoma (R/R LBCL) for Expanded Access and Commercial Out-of-Specification (OOS) Product

Syncytin 1 dependent horizontal transfer of marker genes from retrovirally transduced cells

Retroviral transduction is routinely used to generate cell lines expressing exogenous non-viral genes. Here, we show that human cells transduced to stably express GFP transfer GFP gene to non-transduced cells. This horizontal gene transfer was mediated by a fraction of extracellular membrane vesicles that were released by the transduced cells. These vesicles carried endogenous retroviral envelope protein syncytin 1 and essentially acted as replication-competent retroviruses. The ability to transfer the GFP gene correlated with the levels of syncytin 1 expression in the transduced cells and depended on the fusogenic activity of this protein, substantiating the hypothesis that endogenous syncytin 1 mediates fusion stage in the delivery of extracellular vesicle cargo into target cells. Our findings suggest that testing for replication-competent retroviruses, a routine safety test for transduced cell products in clinical studies, should be also carried out for cell lines generated by retroviral vectors in in vitro studies.

Generation of CAR+ T Lymphocytes Using the Sleeping Beauty Transposon System.

Adoptive immunotherapy of cancer using T cells expressing chimeric antigen receptors (CARs) is now an approved treatment for non-Hodgkin lymphoma (NHL) and B cell acute lymphoblastic leukemia (B-ALL), inducing high response rates in patients. The infusion products are generated by using retro- or lentiviral transduction to induce CAR expression in T cells followed by an in vitro expansion protocol. However, use of viral vectors is cumbersome and is associated with increased costs due to the required high titers, replication-competent retrovirus (RCR) detection and production/use in a biosafety level 2 culture rooms, and additional quality control tests. Nonviral methods, like the Sleeping Beauty transposon system, can stably integrate in the genome of target cells and can be delivered using straightforward methods like electroporation. This chapter describes a protocol for T cell genetic modification using Sleeping Beauty transposon system and electroporation with the Lonza Nucleofector II device for the stable expression of CAR molecules in T lymphocytes.

Retroviral vector production by transient transfection of 293VEC-GALV cells

Background & Aim Introduction Retroviral vectors can permanently integrate their genetic material into chromosomes in the target cells for long-term, stable expression of the introduced genetic elements. It has been widely used since the 1980s and is the second most commonly employed system for gene transfer. Traditionally, retroviral vector production involves the preparation and characterization of a stable producer cell line from which the vector is produced as supernatant. It usually takes about 12 to 20 months from generation the stable producer cell line clone to the final release of the retroviral vector for clinical use. Due to the recent rapid growth of CAR-T cell therapies, a faster, more efficient method for retroviral vector production is required. We have, therefore, employed a novel manufacturing system using transient transfection for rapid GMP preparation of retroviral vectors. Methods, Results & Conclusion Methods Clinical retroviral vectors are produced from a master cell bank of 293Vec-Galv (BioVec Pharma, Quebec, Canada), a human embryonic kidney HEK293-based packaging cell line that stably expresses the Moloney Murine Leukemia gag-pol and Gibbon Ape Leukemia Virus envelope viral proteins. The cells are transfected with plasmid DNA that carries the gene(s) of interest (purified plasmid DNAs are produced by Puresyn Inc. Malvern PA, USA or Aldevron Inc. Fargo ND, USA). The transfection is carried out at 37°C and is mediated by PEIpro®-HQ (Polyplus Transfection, Illkirch, France), which is a highly qualified grade of polyethylenimine developed for large-scale bioproduction processes. About 24 hours post-transfection the medium is replaced by fresh complete medium. Supernatant containing the retrovirus is harvested at 48, 72 and 96 hours post-transfection and filtered through 0.45µm filter. The filtered supernatant is aliquoted, snap frozen and immediately transferred to storage in -80°C freezers. Results and Conclusion Eight retroviral vectors have been manufactured using this novel system at the CAGT GMP Vector Production Facility. Vector production from a single master cell bank of 293Vec-Galv takes about 4 weeks. Four vectors have been released for clinical trials. The transduction rate, phenotype and cytotoxic effect of T cells transduced by vectors produced by this novel system met release requirements, including absence of replication-competent retrovirus. Retroviral vector production by transient transfection of 293Vec-Galv cells saves time, is cost-efficient and safe ( Table 1 ).

Robinow syndrome skeletal phenotypes caused by the WNT5AC83S variant are due to dominant interference with chondrogenesis.

Heterozygous missense mutations in several genes in the WNT5A signaling pathway cause autosomal dominant Robinow syndrome 1 (DRS1). Our objective was to clarify the functional impact of a missense mutation in WNT5A on the skeleton, one of the main affected tissues in RS. We delivered avian replication competent retroviruses (RCAS) containing human wild-type WNT5A (wtWNT5A), WNT5AC83S variant or GFP/AlkPO4 control genes to the chicken embryo limb. Strikingly, WNT5AC83S consistently caused a delay in ossification and bones were more than 50% shorter and 200% wider than controls. In contrast, bone dimensions in wtWNT5A limbs were slightly affected (20% shorter, 25% wider) but ossification occurred on schedule. The dysmorphology of bones was established during cartilage differentiation. Instead of stereotypical stacking of chondrocytes, the WNT5AC83S-infected cartilage was composed of randomly oriented chondrocytes and that had diffuse, rather than concentrated Prickle staining, both signs of disrupted planar cell polarity (PCP) mechanisms. Biochemical assays revealed that C83S variant was able to activate the Jun N-terminal kinase-PCP pathway similar to wtWNT5A; however, the activity of the variant ligand was influenced by receptor availability. Unexpectedly, the C83S change caused a reduction in the amount of protein being synthesized and secreted, compared to wtWNT5A. Thus, in the chicken and human, RS phenotypes are produced from the C83S mutation, even though the variant protein is less abundant than wtWNT5A. We conclude the variant protein has dominant-negative effects on chondrogenesis leading to limb abnormalities.

A Phase I-II Study Using Rexin-G Tumor-Targeted Retrovector Encoding a Dominant-Negative Cyclin G1 Inhibitor for Advanced Pancreatic Cancer

Rexin-G is a replication-incompetent retroviral vector displaying a cryptic SIG-binding peptide for targeting abnormal Signature (SIG) proteins in tumors and encoding a dominant-negative human cyclin G1 construct. Herein we report on the safety and antitumor activity of escalating doses of Rexin-G in gemcitabine-refractory pancreatic adenocarcinoma, with one 10-year survivor. For the safety analysis (n = 20), treatment-related grade 1 adverse events included fatigue (n = 6), chills (n = 2), and headache (n = 1), with no organ damage and no DLT. No patient tested positive for vector-neutralizing antibodies, antibodies to gp70, replication-competent retrovirus (RCR), or vector integration into genomic DNA of peripheral blood lymphocytes (PBLs). For the efficacy analysis (n = 15), one patient achieved a complete response (CR), two patients had a partial response (PR), and 12 had stable disease (SD). Median progression-free survival (PFS) was 2.7, 4.0, and 5.6 months at doses 0–I, II, and III, respectively. Median overall survival (OS) and 1-year OS rate at dose 0–I were 4.3 months and 0%, and at dose II–III they were 9.2 months and 33.3%. To date, one patient is still alive with no evidence of cancer 10 years after the start of Rexin-G treatment. Taken together, these data suggest that Rexin-G, the first targeted gene delivery system, is uniquely safe and exhibits significant antitumor activity, for which the FDA granted fast-track designation.

Screening Clinical Cell Products for Replication Competent Retrovirus: The National Gene Vector Biorepository Experience

Replication-competent retrovirus (RCR) is a safety concern for individuals treated with retroviral gene therapy. RCR detection assays are used to detect RCR in manufactured vector, transduced cell products infused into research subjects, and in the research subjects after treatment. In this study, we reviewed 286 control (n = 4) and transduced cell products (n = 282) screened for RCR in the National Gene Vector Biorepository. The transduced cell samples were submitted from 14 clinical trials. All vector products were previously shown to be negative for RCR prior to use in cell transduction. After transduction, all 282 transduced cell products were negative for RCR. In addition, 241 of the clinical trial participants were also screened for RCR by analyzing peripheral blood at least 1 month after infusion, all of which were also negative for evidence of RCR infection. The majority of vector products used in the clinical trials were generated in the PG13 packaging cell line. The findings suggest that screening of the retroviral vector product generated in PG13 cell line may be sufficient and that further screening of transduced cells does not provide added value.

A Sophism in Vectorology: Turning Harmful Defective Retroviral Vectors into Helpful Replication-Competent Retroviruses Against Cancer.

A Sophism in Vectorology: Turning Harmful Defective Retroviral Vectors into Helpful Replication-Competent Retroviruses Against Cancer.

Stable knockdown of CREB, HIF-1 and HIF-2 by replication-competent retroviruses abrogates the responses to hypoxia in hepatocellular carcinoma

The fast proliferation of tumor cells develops faster than the vasculature, resulting, in most malignant tumors, in generation of hypoxic regions. Hypoxia renders solid tumors resistant to radiation and chemotherapeutics while providing opportunities for tumor-selective therapies targeting tumor hypoxia. Here we exploit two properties of tumors: propagation of tumor cells and ongoing generation of hypoxic regions to construct a system that preferentially leads to the death of tumor cells and thus hinders tumor growth. We constructed murine leukemia virus replication-competent (RCR) viruses that infect only propagating cells. These viruses express small hairpin RNAs (shRNAs) targeting cyclic AMP-response-element binding protein (CREB), hypoxia-inducible factors 1 (HIF)-1 or HIF-2 individually or all three together (X3). These viruses efficiently infected in vitro human hepatocellular carcinoma (HepG2 and FLC4) cells and established persistence of the virus and knocked down the expression of the regulators of the hypoxia-responding genes. Knockdown of either HIF-1 or CREB or both in hypoxia reduced the expression of hypoxia-response elements- and CRE-mediated gene expression, diminished cell proliferation and increased caspase-3 activity. We did not detect any significant effect of the efficiently knocked down HIF-2 on any of the functions tested in vitro. Moreover, severe combined immunodeficiency mice implanted subcutaneously with HepG2 stably infected with recombinant RCRs showed reduction of tumor growth and vascular endothelial growth factor expression, and no hypoxia-guided neovascularization. Combined treatment (RCRs+doxorubicin) improved efficacy in the context of in vitro hypoxia and in vivo (with either vACE-CREB or vACE-X3). This synergistic effect may lead to an improved efficacy and safety profile of the treatment that may result in fewer side effects.

Safety Data from a First-in-Human Phase 1 Trial of NKG2D Chimeric Antigen Receptor-T Cells in AML/MDS and Multiple Myeloma

Introduction: Conventional CAR-T cells express a single chain antibody variable fragment that restricts recognition to one tumor antigen and a limited set of cancers. This study employs a novel CAR fusing full-length human NKG2D with the CD3z signaling domain. In autologous transduced CM-CS1 T cells, NKG2D CAR receives endogenous costimulation via DAP10 to target multiple NKG2D-ligands that are upregulated in solid and hematologic malignancies but absent or poorly expressed on healthy tissues. Methods: A phase 1 dose-escalation study to establish safety and feasibility of a single infusion of CM-CS1 T cells without lymphodepleting conditioning enrolled subjects with AML/MDS-RAEB or relapsed/refractory progressive multiple myeloma (MM) without standard therapy options (NCT02203825). Eligibility criteria included suitable organ function, no CNS disease, no prior allogeneic SCT or adoptive T-cell therapy, no therapy within 3 weeks prior to infusion, no immune suppression, and no uncontrolled infection. Dose-escalation spanned 4 cohorts [half-log increments from 1x106 to 3x107 CM-CS1 T cells] according to a 3+3 design. DLTs included ≥ Grade 3 non-hematologic toxicity or ≥ Grade 2 autoimmune toxicity related to CAR T cells. Initial assessment was at 28 days. At least 1 AML/MDS and 1 MM subject were mandated in each dose level. Manufacturing included PBMC stimulation with OKT3 and IL-2 followed by 2 rounds of retroviral transduction at DFCI9s Cell Manipulation Core Facility. Vector copy number (VCN) and replication-competent retrovirus (RCR) testing were performed on whole blood and PBMCs, respectively, using quantitative PCR. Results: From April 2015 to July 2016, 11 subjects were infused, and 10 completed the DLT period. Eight of 11 were male, 6 had AML/MDS, and median age was 70 (range 44 to 79) (Panel A). Median WBC was 2.3 (range 0.7 to 7.2 K/uL); median ALC was 0.74 (range 0.09-2.37 K/uL). Five had cells manufactured from peripheral blood; 6 underwent apheresis. Median percentage of blasts in bone marrow for AML/MDS patients was 50% (range 4-68%). All myeloma patients had undergone ≥ 5 therapies including ≥1 autologous SCT. Four of the 6 AML/MDS patients had secondary disease, 3 had complex cytogenetics, 3 had p53 mutations, and 1 had a FLT3-ITD mutation. Dose-escalation proceeded from 1x106 to 3x107 CM-CS1 T cells. All 11 products passed release criteria, and there were no infusion reactions. Products consisted of median 97.2% CD3+ cells and 31.0% CD8+ cells, with vector-specific NKG2D expression on median 74.6% of CD3+ and 66.3% of CD8+ cells (Panel B). The first 10 subjects completed their 28 day evaluation period without DLTs. There were no cases of cytokine release syndrome, cell-related neurotoxicity, auto-immunity, or CAR T-related death. SAEs included a Grade 4 intracochlear bleed and an episode each of grade 4 neutropenia and thrombocytopenia deemed related to disease progression. Forty percent of patients experienced some Grade 3 toxicity, all related to underlying disease or a complication thereof (Panel C). At these initial cell doses, no patient to date has had objective tumor response at the 28 day evaluation mark. Nine initiated subsequent therapies; there have been 4 deaths secondary to disease or complications of subsequent therapies. However, cases of unexpected survival without further therapy and responses to subsequent treatments were noted. For example a patient with p53-mutated AML survived 4 months despite 50% blasts at infusion, and another entered PR at 6+months after cells on an IDH-1 inhibitor with Conclusion: In the first 3+ dose-escalation cohorts of patients with AML/MDS and myeloma, a single dose of CM-CS1 T cells without lymphodepletion was feasible and well-tolerated, with no DLTs. CAR T cells generally have not persisted beyond 1 week, consistent with pre-clinical models. Correlative analyses including post-infusion immunophenotyping are in process. Future studies of multiple infusions of NKG2D CAR T cells in both hematologic malignancies and solid tumors at the higher cell doses associated with efficacy in pre-clinical models are in planning. Disclosures Murad:Celdara Medical, LLC: Employment. Reder:Celdara Medical, LLC: Employment. Sentman:Celdara Medical, LLC: Membership on an entity9s Board of Directors or advisory committees, Other: Holds patents on this technology. Wade:Celdara Medical, LLC: Employment. Schmucker:Celdara Medical, LLC: Employment. Lehmann:Celyad, SA: Employment. Snykers:Celyad, SA: Employment. Allen:Celyad, SA: Employment. Stone:Celator: Consultancy; Jansen: Consultancy; Novartis: Consultancy; Merck: Consultancy; ONO: Consultancy; Sunesis Pharmaceuticals: Consultancy; Roche: Consultancy; Celgene: Consultancy, Membership on an entity9s Board of Directors or advisory committees; Juno Therapeutics: Consultancy; Xenetic Biosciences: Consultancy; Agios: Consultancy; Abbvie: Consultancy, Membership on an entity9s Board of Directors or advisory committees; Seattle Genetics: Consultancy; Pfizer: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy. Soiffer:Kiadis: Membership on an entity9s Board of Directors or advisory committees; Juno: Consultancy. Dranoff:Novartis: Employment. Ritz:Kiadis: Membership on an entity9s Board of Directors or advisory committees.

Long-Term Safety Follow-Up of Subjects Previously Treated with Non-Replicating Retroviral Vector-Based Gene Therapies

ObjectiveOur objective was to evaluate the life-long safety profile of gene therapy using retroviral (non-replicating) vectors (nRCR), or cell products in 127 subjects with hemophilia, human immunodeficiency virus (HIV), or cancer, previously treated with such gene therapy.MethodsWe assessed the occurrence of serious adverse events (SAEs), deaths and presence of replication competent retrovirus (RCR).ResultsA total of 23 subjects remained until the data cut-off date of 31 July 2013 and provided safety information of up to 18 years. Of the 104 subjects who discontinued, the primary reason was loss to follow-up (47.2 %; n = 60). The follow-up period for the 60 subjects lost to follow-up was 7–10 years. A total of 41 subjects experienced at least one SAE, and 15 subjects died. We reviewed SAEs and cause of death (none related to the active therapy), but no evidence was found for safety signals related to new malignancy or neurologic, rheumatological, autoimmune, or hematologic disorder. RCR results were negative, indicating no evidence for in vivo vector persistence.ConclusionDespite the loss of follow-up, which is the limiting factor in this long-term safety trial, the findings from this long-term follow-up study are encouraging.

71. Validation of Manufacturing NKG2D Chimeric Antigen Receptor T Cells for a First-In-Human Clinical Trial in AML/MDS and Multiple Myeloma

Canonical CAR-T cells are engineered using constructs encoding a single chain variable fragment derived from a murine or humanized antibody, a costimulatory domain connected by a hinge region, and the signaling domain of CD3 zeta. This restricts T cells transduced with these constructs to recognizing one tumor antigen, such as CD19, and a limited set of cancers. Prior work in murine models demonstrated preclinical efficacy of T cells transduced with a novel activating CAR construct that exploits the ability of NK cells to recognize a variety of tumor antigens. Specifically, a CAR was generated by fusion of native full-length human Natural Killer Group 2D (NKG2D) gene with the human CD3 zeta cytoplasmic signaling domain. The NKG2D receptor in association with natural costimulatory molecule DAP10 creates a unique 6 protein receptor complex enabling NK and CD8+ T cells to kill many cells types via recognition of ligands including MIC-A, MIC-B, and UL-16 binding proteins 1-6. NKG2D-ligands are upregulated in malignancy but protein expression is absent or minor in healthy tissues. Complete remissions and durable CD4+ and CD8+ T-cell memory have been demonstrated in murine models of lymphoma, myeloma and ovarian cancer following adoptive therapy with NKG2D CAR T cells. At the same time, NKG2D CAR T cells significantly alter the tumor microenvironment through cytokine secretion to further promote anti-tumor immunity. This technology is being evaluated clinically in a phase I dose-escalation study (ClinicalTrials. gov NCT02203825) arising through collaboration between academic investigators and commercial sponsors. GMP manufacturing procedures for NKG2D CAR T cells were developed to support this first-in-human trial of NKG2D CAR T cells to assess safety and feasibility in acute myeloid leukemia/myelodysplastic syndrome and multiple myeloma. Following isolation of mononuclear cells, T cells were activated with anti-CD3 mAb and IL-2, subjected to 2 rounds of transduction with SFG retroviral vector containing the NKG2D CAR construct (CM-CS1), and expanded in media containing IL-2. GMP manufacturing procedures were validated using T cells from healthy donors as well as patients with AML and myeloma. Validation studies and initial clinical manufacturing demonstrated consistent viability, robust cell expansion, vector-mediated surface expression of NKG2D on a median of 53.6% CD8+ and 90.3% of CD4+ T cells, consistent viral copy number/cell less than 5, and the absence of replication-competent retrovirus in CM-CS1 T cells. Functionally, CM-CS1 T cells exhibit potent IFN-γ production following exposure to NKG2D ligand-expressing tumor cells. We also demonstrated minimal carry-through of malignant myeloid or plasma cells after deriving autologous CM-CS1 T cells from patients with malignancies. The Phase I trial using CM-CS1 T cells is ongoing, with dose-escalation proceeding as planned. Future goals include application of this novel cell therapy to additional malignant diseases.

Tissuegene-C (INVOSSA™) in patients with osteoarthritis: A phase II trials

Purpose: TissueGene-C (TG-C) is a cell mediated gene therapy that contains non-transduced (hChonJ) and transduced (hChonJb#7) human allogeneic chondrocytes. The hChonJb#7 cells were transduced with TGF-β1 gene by using retroviral vector and irradiated with gamma-ray. TG-C has been demonstrated its efficacy and safety in preclinical studies in vitro and in vivo. TG-C has been proven for its safety and efficacy in Phase I and Phase IIa clinical studies in osteoarthritis (OA) patients. The current placebo controlled phase IIb study was conducted to determine both safety and efficacy of TG-C in patients with OA of the knee. Methods: Participants (n = 54) with a confirmed diagnosis of knee OA by X-ray and MRI were randomized into the treatment group (TG-C, 1.8x107cells/knee, n = 27) and the placebo group (saline, n = 27). The primary evaluation parameter was International Knee Documentation Committee (IKDC) which measures pain, sports activities, and daily function. The secondary evaluation parameters were Western-Ontario and MacMaster University (WOMAC) score, Knee Injury and Osteoarthritis Outcome Score (KOOS), and 100 mm Visual Analogue Scale (VAS). These parameters were assessed at 12 and 24 weeks post treatment. For TG-C treatment group, the evaluation was extended to 48 weeks post treatment. Additionally, changes in biomarkers were assessed in serum and urine samples. Safety measures, including physical exams, complete blood count, and serum chemistry were included up to 6 months post treatment. Blood samples were screened to detect the replication competent retrovirus (RCR), retrovirally transduced cells, and TGF-β1 DNA and protein starting from 2 weeks up to 6 months post treatment. Results: TG-C treatment group showed improvement in IKDC, WOMAC, KOOS and 100 mm VAS scores compared to placebo group, which was maintained up to the 1 year. Conclusions: In summary, the Phase IIb study indicated that TG-C (InvossaTM) treatment improved pain, sports activities, and quality of daily life in patients with knee OA when compared to the placebo control.

Biochemical properties of the xenotropic murine leukemia virus-related virus integrase.

Xenotropic Murine Leukemia Virus-related Virus (XMRV) is a new gammaretrovirus generated by genetic recombination between two murine endogenous retroviruses, PreXMRV1 and PreXMRV2, during passaging of human prostate cancer xenografts in laboratory mice. XMRV is representative of an early founder virus that jumps species from mouse to human cell lines. Relatively little information is available concerning the XMRV integrase (IN), an enzyme that catalyzes a key stage in the retroviral cycle, and whose sequence is conserved among replication competent retroviruses emerging from recombination between the murine endogenous PreXMRV-1 and PreXMRV-2 genomes. Previous studies have shown that IN inhibitors efficiently block XMRV multiplication in cells. We thus aimed at characterizing the biochemical properties and sensitivity of the XMRV IN to the raltegravir, dolutegravir, 118-D-24 and elvitegravir inhibitors invitro. We report for the first time the purification and enzymatic characterization of recombinant XMRV IN. This IN, produced in Escherichia coli and purified under native conditions, is optimally active over a pH range of 7-8.5, in the presence of Mg(2+) (15mM and 30mM for 3'-processing and strand transfer, respectively) and is poorly sensitive to the addition of dithiothreitol. Raltegravir was shown to be a very potent inhibitor (IC50∼30nM) whereas dolutegravir and elvitegravir were less effective (IC50∼230nM and 650nM, respectively). The 118-D-24 drug had no impact on XMRV IN activity. Interestingly, the substrate specificity of XMRV IN seems to be less marked compared to HIV-1 IN since XMRV IN is able to process various donor substrates that share little homology. Finally, our analysis revealed some original properties of the XMRV IN such as its relatively low sequence specificity.

Tissuegene-C (TG-C) improved clinical scores in patients with osteoarthritis: a phase 2B study

s / Osteoarthritis and Cartilage 22 (2014) S57–S489 S194 333 TISSUEGENE-C (TG-C) IMPROVED CLINICAL SCORES IN PATIENTS WITH OSTEOARTHRITIS: A PHASE 2B STUDY B. Lee, J. Cho, T. Kim, Y. Park, E. Jeong, K.-H. Lee. Kolon Life Sci., Inc., Gwacheon-si, Republic of Korea Purpose: TG-C is a cell mediated gene therapy that contains nontransduced (hChonJ) and transduced (hChonJb#7) human allogeneic chondrocytes. The hChonJb#7 cells were transduced with TGF-b1 gene by using retroviral vector and irradiated with gamma-ray. TG-C has been proved its efficacy and safety in preclinical studies in vitro and in vivo. TG-C has been tested in Phase I and Phase IIa clinical studies in osteoarthritis (OA) patients and has proved its safety and efficacy in the patients. Methods: The current placebo controlled phase IIb study was conducted to determine both safety and efficacy of TG-C in patients with OA of the knee. Participants (n1⁄4 54) with a confirmed diagnosis of knee OA by X-ray and MRI were randomized into the treatment group (TG-C, 1.8 107 cells/knee, n 1⁄4 27) and the placebo group (saline, n 1⁄4 27). The primary evaluation parameter was International Knee Documentation Committee (IKDC) which measures pain, sports activities, and daily function. The secondary evaluation parameters were Western-Ontario and MacMaster University (WOMAC) score, Knee Injury and Osteoarthritis Outcome Score (KOOS), and 100 mm Visual Analogue Scale (VAS). These parameters were assessed at 12 and 24 weeks post treatment. For TG-C treatment group, we also evaluated the all evaluation parameters at 48 weeks post treatment. Additionally, changes in biomarkers were assessed in serum and urine samples. Safety measures, including physical exams, complete blood count, and serum chemistry were included up to 6 months post treatment. Blood samples were screened to detect the replication competent retrovirus (RCR), retrovirally transduced cells, and TGF-b1 DNA and protein starting from 2 weeks up to 6 months post treatment. Results: TG-C treatmentgroup showed improvement in IKDC, WOMAC, KOOS and 100 mm VAS scores compared toplacebo group as shown in Table 1.

Biosafety Challenges for Use of Lentiviral Vectors in Gene Therapy

Lentiviral vectors are promising tools for the genetic modification of cells in biomedical research and gene therapy. Their use in recent clinical trials for the treatment of adrenoleukodystrophy, β-thalassemia, Wiskott-Aldrich- Syndrome and metachromatic leukodystrophy underlined their efficacy for therapies especially in case of hereditary diseases. In comparison to gammaretroviral LTR-driven vectors, which were employed in the first clinical trials, lentiviral vectors present with some favorable features like the ability to transduce also non-dividing cells and a potentially safer insertion profile. However, genetic modification with viral vectors in general and stable integration of the therapeutic gene into the host cell genome bear concerns with respect to different levels of personal or environmental safety. Among them, insertional mutagenesis by enhancer mediated dysregulation of neighboring genes or aberrant splicing is still the biggest concern. However, also risks like immunogenicity of vector particles, the phenotoxicity of the transgene and potential vertical or horizontal transmission by replication competent retroviruses need to be taken into account. This review will give an overview on biosafety aspects that are relevant to the use of lentiviral vectors for genetic modification and gene therapy. Furthermore, assay systems aiming at evaluating biosafety in preclinical settings and recent promising clinical trials including efforts of monitoring of patients after gene therapy will be discussed.

Production of Lentivectors for Clinical Use

Since the first human gene therapy clinical trials were proven successful in 2000, human gene therapy has witnessed setbacks as well as encouraging progress. While these first clinical trials were performed with a γ-retrovirus vector to correct X-linked severe combined immunodeficiency (SCID-X1) in children, others since then have applied similar or alternative viral vector strategies. More recently, lentiviral vectors have been used for the correction of human β- thalassaemia and adrenoleukodystrophy. They have also been successfully used in the treatment of leukaemia by modifying cytotoxic T cells directed to cancer cells. Here we describe the production of clinical grade retro- and lentiviral vectors for application in human therapy. Keywords: Biosafety, clinical grade lentivector, gene therapy, genotoxicity, GMP guidelines, HIV, immunology, insertional mutagenesis, mutagenesis assay, packaging cell lines, purification, replication competent lentivirus, replication competent retrovirus.