Self-inactivating Vector Research Articles

Preclinical Assessment for Gene Addition Strategy in Familial Hemophagocytic Lymphohistiocytosis Related to Munc 13-4 Deficiency

Introduction: Familial Hemophagocytic Lymphohistiocytosis (FHL) comprises a group of rare genetic disorders with defect cytotoxicity in T and NK cells predisposing to severe hyperinflammation with the clinical hallmark of the Hemophagocytic LymphoHistiocytosis (HLH) syndrome. The uncontrolled proliferation of activated T-CD8 lymphocytes and macrophages infiltrates lymphoid tissues, bone marrow, and multiple organs including the brain. Thus, if untreated full-scale FHL is usually fatal. FHL3 is due to mutations in the UNC13D gene coding for Munc 13-4 protein, one of the components of the perforin-dependent cytotoxicity apparatus. This condition accounts for 30% to 35 % of all genetic cases of HLH. Hematopoietic stem and progenitor cell (HSPC) transplantation, which is the only curative treatment for FHL3 to date, remains difficult even when a compatible donor is available because of the patients' critical inflammatory background. In this context, gene therapy could be a promising therapeutic option, especially for those patients without HLA-compatible donor. Method: The feasibility and efficacy of gene addition strategy for FHL3 have been previously demonstrated in mouse models. We then translated this approach to human cells using a third-generation self-inactivated lentivirus vector pseudo-typed with a conventional vesicular stomatitis virus-G (VSVG) expressing a codon-optimized UNC13D gene under the transcriptional control of EF1 alpha (LV-EF1a-UNC13D). HSPC or CD3+ T-cells were transduced with a GMP-like-grade LV-EF1a-UNC13D lentiviral vector. We studied the restoration of cytotoxic function. Results: Our GMP transduction protocol led to efficient transcription of optimized mRNA codon and expression of Munc 13-4 protein as evidenced by RT qPCR and western blot, respectively. Interestingly, we show restored cytotoxicity activity after CD34+ differentiation toward CD3+ T-cells and NK cells. The vector copy number is compatible with a clinical approach. In vivo experiments are ongoing to validate the absence of genotoxicity in this setting. Discussion conclusion: Our results support the feasibility and efficacy of UNCD13D gene transfer into hematopoietic stem and progenitor cells or T cells from Munc 13-4 deficient patients. Alternatively, gene therapy of HSPCs could be combined with transduced autologous T-cells infusion. The first attempts regarding this option are encouraging. UNC13D gene addition strategy could dramatically improve the care of FHL3 patients lacking an HLA-genoidentical donor.

Efficient Chimeric Antigen Receptor T-Cell Generation Starting with Leukoreduction System Chambers of Thrombocyte Apheresis Sets

Introduction: Primary human blood cells represent an essential model system to study physiology and disease. However, human blood is a limited resource. During healthy donor plateletpheresis, the leukoreduction system chamber (LRSC) reduces the leukocyte amount within the subsequent platelet concentrate through saturated, fluidized, particle bed filtration technology. Normally, the LRSC is discarded after apheresis is completed. Compared to peripheral blood, LRSC yields 10-fold mononuclear cell concentration. Methods: To explore if those retained leukocytes are attractive for research purposes, we isolated CD3+ T cells from the usually discarded LRSCs via density gradient centrifugation in order to manufacture CD19-targeted chimeric antigen receptor (CAR) T cells. Results: Immunophenotypic characterization revealed viable and normal CD4+ and CD8+ T-cell populations within LRSC, with low CD19+ B cell counts. Magnetic-activated cell sorting (MACS) purified CD3+ T cells were transduced with CD19 CAR-encoding lentiviral self-inactivating vectors using concentrated viral supernatants. Robust CD19 CAR cell surface expression on transduced T cells was confirmed by flow cytometry. CD19 CAR T cells were further enriched through anti-CAR MACS, yielding 80% CAR+ T-cell populations. In vitro CAR T cell expansion to clinically relevant numbers was achieved. To prove functionality, CAR T cells were co-incubated with the human CD19+ B cell precursor leukemia cell line Nalm6. Compared to unmodified T cells, CD19 CAR T cells effectively eradicated Nalm6 cells. Conclusion: Taken together, we can show that lymphocytes isolated from LRSCs of plateletpheresis sets can be efficiently used for the generation of functional CAR T cells for experimental purposes.

Long-Term Outcome of Gene Therapy for X-Linked Severe Combined Immunodeficiency (SCID-X1) Using an Enhancer-Deleted Self-Inactivating Gammaretroviral Vector

Long-Term Outcome of Gene Therapy for X-Linked Severe Combined Immunodeficiency (SCID-X1) Using an Enhancer-Deleted Self-Inactivating Gammaretroviral Vector

Lentiviral Gene Therapy with Low Dose Conditioning for X-Linked SCID Results in Complete Immune Reconstitution and No Evidence of Clonal Expansion

Introduction: Gene therapy (GT) for X-linked severe combined immunodeficiency (SCID-X1) using a gamma (γ) retroviral vector resulted in robust T cell immune reconstitution but also insertional oncogenesis. A multi-institutional trial using a self-inactivating (SIN) γ-retroviral vector resulted in equivalent T cell immune reconstitution but no insertional oncogenesis to date (Hacein-Bey-Abina et al, NEJM 2014). In both trials, conditioning was only rarely used and therefore gene marking in B cells and neutrophils was negligible and reconstitution of NK cells was limited. To improve both the immune reconstitution and safety of GT for SCID-X1, we developed a lentiviral vector (LV) in which a codon optimized IL2RG transgene is driven by the elongation factor 1 alpha short promoter (EFS) and introduced low dose busulfan conditioning to enhance hematopoietic and multilineage expression of IL2RG. An international, multicenter, Phase 1/2 clinical trial (NCT03311503, NCT03601286) is currently underway to evaluate the safety and efficacy of LV mediated gene transfer using this vector. Methods: A total of 14 patients have been enrolled. Two patients were withdrawn due to poor stem cell mobilization and collection and manufacturing are in progress for 3 patients. Nine patients have been infused, 8 receiving mobilized peripheral blood stem cells and 1 receiving bone marrow at an average age of 5.3 months (range 3.2-7.1). Hematopoietic stem cells (CD34+ cells) were transduced with the LV vector (using transduction enhancers in 7 products) and drug products were cryopreserved. Median cell dose was 9.06 x 10e6 CD34+ cells/kg (4.55-17.87) and median vector copy number (VCN) was 0.86 copies/diploid genome(dg) (0.67-2.79). Drug products were thawed and infused after 2 days of busulfan conditioning targeted to an AUC of 30 mg*h/L. All infants tolerated busulfan exposure without any significant toxicity. Results: As of July 2022, all treated patients are alive with robust reconstitution of gene marked T cells with median follow up of 2.2 years (0.2-4.3). T cell reconstitution was rapid with median CD3+ T cell count 552 cells/uL (132-1160) at 3 months. Seven patients evaluable at 1 year had CD3+ (2377-4900 cells/uL) and CD4+ T cell counts (1507-3080 cells/uL) normal for age at 1-year post-GT (Figure 1). Gene marking in T cells was robust averaging 1.55-5.66 copies/dg. Evidence of thymic function includes rising naïve CD4+ T cells and maintenance of a normal CD4/CD8 ratio. Multilineage engraftment of gene modified cells is evident in B cells, neutrophils, and NK cells at last follow-up with stable VCN over time (ranges 0.43-2.05, 0.24-2.11, 1.98-3.61 respectively). In contrast to limited NK cell reconstitution seen in the absence of conditioning in prior trials, NK cell counts have been sustained in 6 of 7 patients with greater than 12 months of follow-up achieving values within the normal range for age (Figure 1). Six of these 7 patients have been able to stop immunoglobulin replacement, 4 of 4 who have completed a primary vaccine series show response, with the results of 2 further patients awaited. Insertion site analysis shows uniformly polyclonal repertoire with no evidence of clonal expansion. Conclusion: In summary, gene therapy using a lentiviral vector and low dose busulfan conditioning resulted in robust and complete immune reconstitution correcting T, NK, and B cell defects characteristic of SCID-X1 with 100% survival and no gene therapy related adverse events. Figure 1 Immune reconstitution in 7 patients treated with >1 year follow-up A) CD3+ T cells. B) CD4+ T cells. C) NK cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immune Reconstitution and Preliminary Safety Analysis Of 9 Patients Treated With Somatic Gene Therapy For X-Linked Severe Combined Immunodeficiency (SCID-X1) With a Self-Inactivating Gammaretroviral Vector

▪X-linked severe combined immunodeficiency (SCID-X1) is a fatal genetic disease currently treated with hematopoietic stem cell transplantation. Previous gene therapy trials using a MLV-based gammaretroviral vector expressing the IL-2 receptor gamma chain (γc) controlled by the viral long-terminal repeat (LTR) induced T cell reconstitution in 18 of 20 boys with SCID-X1, but also resulted in insertional oncogenesis in 5 of 20, leading to leukemia, with 4 of the 5 demonstrating insertion at the LMO2 locus (Trial 1). We tested whether a self-inactivating (SIN) gammaretroviral vector, pSRS11.EFS.IL2RG.pre*, derived by removing the MLV LTR U3 (enhancer/promoter) region and driving expression of γc by an intronless elongation factor 1α (EF1-α) promoter, would promote T cell reconstitution in boys with X-SCID with an improved safety profile (Trial 2). Pre-clinical studies showed a lack of clonal skewing in primary and secondary transplant recipient mice in vivo and lack of insertions in either Lmo2 or Evi1 in these mice. The primary endpoints of the study are: 1. immune recovery defined as CD3+ T cell count >300/microliter and restoration of proliferation to phytohemagglutinin (PHA); and 2. Lack of insertional oncogenesis.To date 9 boys with SCID-X1 age 3.9 to 10.5 months were enrolled with 5-32 months follow-up on the current trial. All received bone marrow derived CD34+ cells transduced with the pSRS11.EFS.IL2RG.pre* vector, infused without conditioning. Transduction was performed at each site with one of two common protocols and the same GMP-produced vector supernatant. Transduction efficiency of infused CD34+ cells ranged from 0.25-2.92 vector copy number (VCN). One patient with a low initial transduction underwent a repeat procedure 1 month after infusion of the first product. Overall survival is excellent with 8 out of 9 patients alive and well; 1 patient died of overwhelming adenoviral infection, present at study entry, at 4 months prior to full reconstitution with genetically modified T cells. Excluding 1 patient with high level maternal engraftment who is unevaluable with regard to T cell number, 5 of 7 (71%) achieved CD3+ T cell count >300/microliter. The 2 patients who did not achieve T cell count >300/microliter received CD34+ cells with the lowest vector copy number (VCN). An early rise in CD16/56+ NK cells was also associated with T cell recovery. Of 8 surviving patients, 7 of 8 (87.5%) had PHA stimulation index >15 at or before 6 months (n=8; median 85.5; range 0.2-224); the patient who had no functional recovery received mismatched allogeneic cord blood transplant. At last follow-up, the remaining 7 patients have evidence of γc transgene expression in T cells, naïve T cell generation, and normal diversity. They are free of SCID related infections and humoral immune evaluation is ongoing.We compared the early immune recovery and peripheral blood insertion site analysis between 6 evaluable subjects on Trial 2 with the 20 subjects enrolled on the previous trials of MLV-based γc vector. The CD3+ T cell count at 6 months for Trial 2 (n=6; median 548/microliter; range 87-3960) does not appear to be lower than for Trial 1 (n=19; median 1755/microliter; range 200-4759; p=0.14 in 2-sided Wilcoxon rank sum test). However, we recognize that this test is underpowered, and results are preliminary. Because the latency time to leukemic events in the previous trial was long (33-60 months), we compared insertion sites as a surrogate measure of safety. Insertional activation of growth controlling genes has been demonstrated to be associated in some murine models with clonal skewing during in vitro expansion and in vivo reconstitution. Therefore, we hypothesized that removal of the transcription factor binding site-rich LTR U3 enhancer regions would change the insertion pattern detected in peripheral blood after transplantation away from the 5' ends of genes important for cell proliferation or persistence. Whereas in Trial 1, LMO2, EVI1 and other lymphoid proto-oncogenes showed prominent clusters of integration sites post-transplantation, these locations were not significant clusters in the second trial (p<10-4; comparison of n=19 Trial 1 versus n=6 Trial 2). Thus, we show promising early data that a modified gammaretroviral vector retains efficacy in treatment of SCID-X1 and is likely to have an improved safety profile. Disclosures:Off Label Use: Off-label use of CliniMACS device for purification of CD34+ bone marrow cells. Baum:Transatlantic Gene Therapy Consortium: Patents & Royalties.

Interim Results from a Phase I/II Clinical Gene Therapy Study for Newly Diagnosed Infants with X-Linked Severe Combined Immunodeficiency Using a Safety-Modified Lentiviral Vector and Targeted Reduced Exposure to Busulfan

▪Early trials of gene therapy for X-linked Severe Combined Immunodeficiency (XSCID) restored T cell immunity in most cases, but did not correct B cell function and carried a high risk of iatrogenic leukemia. The subsequent development of self-inactivating γ-retroviral vectors has enhanced safety, but has not restored B cell function to date. We developed a new approach for XSCID gene therapy that utilizes a safety modified lentiviral (LV) vector (CL20-i4-EF1α-hGC-OPT) together with reduced exposure busulfan (Bu) conditioning for newly diagnosed infants with XSCID. Recently, we reported that the combination of reduced dose Bu used together with our LV vector restored T and B cell function in older XSCID children with declining immune function following haploidentical transplantation (De Ravin SS et al, Sci Transl Med, 2016). Here we report initial results of LVXSCID-ND; a multi-center, phase I/ II safety and efficacy study using our LV vector and dose-adjusted Bu for the first time in newly diagnosed XSCID infants. We enrolled six subjects over the last 12 months (median age of 4.5 months, range: 2-12 months). Purified bone marrow (BM) CD34+ cells were transduced with the CL20-i4-EF1α-hGC-OPT vector generated by a stable producer cell line and then cryopreserved to facilitate central manufacturing for multiple study sites and evaluation of release criteria prior to conditioning. Busulfan was given as two single daily doses to target a total cumulative area-under-the-curve (cAUC) of 22 mg*hr/L (60-90 mg*hr/L = myeloablative cAUC). The median dose of transduced CD34+ cells was 8.06 x 106 cells/kg (range: 4.6 - 11 x 106) and the median vector copy number (VCN) in graft CFU-C was 0.40 copies /cell (range: 0.16 - 0.97). An average Bu cAUC of 22.9 mg*hr/L (range: 20.0 to 24.2) was achieved, which was within 10% of the intended cAUC in all patients. As of July 2017, no severe adverse events related to BM harvest, Bu exposure, or cell infusion have been observed. In the first 5 evaluable cases, complete hematopoietic recovery occurred by 3-4 weeks without any blood product support. Follow-up data from the oldest patient who presented with high levels of maternal T cell engraftment, severe neutropenia requiring G-CSF therapy, CMV viral infection, with a graft VCN of 0.16, and who is now 12 months post therapy, demonstrated delayed and partial T cell reconstitution. Cases 2 and 3 have been followed for nine and six months, respectively and have significantly higher VCNs in peripheral blood (PB) subsets (CD14/15+myeloid cells 0.65, 0.30 copies/cell: CD3+ T cells 2.78, 2.71; CD19+ B cells 1.01, 0.78; and CD56+ NK cells 2.72, 2.11 respectively). Bone marrow aspirates on week 16 yielded VCNs in sorted CD34+ cells of 0.56, 0.50; and BM myeloid CFU-GM of 0.61, 0.24. Rapid T cell reconstitution in cases 2 and 3 resulted in normal numbers of CD3+, CD4+, CD4 naïve, and CD8+ cells with TRECs 626 and 1170 copies per ug DNA 16-20 weeks post therapy. In both cases at 16 weeks, T cell proliferation was 86% and 81% of control, and V-β spectratype scores were normal at 194 and 155. At nine months, case 2 had a normal isohemagglutinin anti-A titer of 1:32 and normal 4-week trough serum immunoglobulins levels (IgG 713 mg/dl, IgM 54 mg/dl, and IgA 16 mg/dl). IVIG supplementation has been discontinued for approximately 3 months before vaccination responses are assessed. Case 4 has been followed for 12 weeks with PB VCNs for CD3+ cells of 1.36 copies/cell, CD19+ 0.58, CD56+ 1.38 and CD14/15+ 0.02. Flow cytometry analysis of PB at 12 weeks shows 4.5% of PB leukocytes are CD3+, with 61% CD4+, 11% CD8+, and a significant fraction expressing a CD45RA+, RO- naive phenotype at this early time point. Cases 5 and 6 are still too early to evaluate for immune phenotype and function. Preliminary vector insertion site analysis shows highly polyclonal marking patterns in case 2 with 11,000 insertion sites in CD3+ cells, 5049 sites in CD19+ cells, 756 sites in BM myeloid CFU-C without any evidence of clonal dominance. In summary, gene therapy for newly diagnosed XSCID patients using a LV vector with targeted reduced exposure Bu conditioning is well tolerated and results in rapid T cell reconstitution in most cases. Efficient vector marking in bone marrow CD34+ cells, myeloid cells, and B cells indicate that this approach will likely provide broad immune reconstitution rather than restricted T cell correction seen in past trials using γ-retroviral vectors with no Bu. DisclosuresLong-Boyle:InsightRX: Consultancy. Puck:UpToDate: Patents & Royalties: Recieve royalties to write and edit entries on primary immune disorders; InVitae, a clinical DNA sequencing company: Other: Spouse employment and stock options. Cowan:Homology Medicine: Consultancy.

PSIR-bsr, a self-inactivating retrovirus vector expressing the blasticidin S-resistance gene

Self-inactivating retrovirus vectors are useful tools for generating stable cell lines harbouring designed exogenous sequences but lacking the constitutive transcriptional activity of the long terminal repeats that are usually retained by non-self-inactivating retrovirus vectors. Thus, self-inactivating retrovirus vectors are ideal vehicles for integrated transgenes comprising transcriptional regulatory sequences, and for the genes expressed by these regulatory sequences. This article describes the development of a self-inactivating retrovirus vector retaining a blasticidin S-resistance (bsr) gene. The vector, named pSIR-bsr, would be useful for transducing multiple expression vectors with different selection markers.

Phase I/II Manufacture of Lentiviral Vectors Under GMP in an Academic Setting.

In clinical gene transfer applications, lentiviral vectors (LV) have rapidly become the primary means to achieve permanent and stable expression of a gene of interest or alteration of gene expression in target cells. This status can be attributed primarily to the ability of the LV to (1) transduce dividing as well as quiescent cells, (2) restrict or expand tropism through envelope pseudo-typing, and (3) regulate gene expression within different cell lineages through internal promoter selection. Recent progress in viral vector design such as the elimination of unnecessary viral elements, split packaging, and self-inactivating vectors has established a significant safety profile for these vectors. The level of GMP compliance required for the manufacture of LV is dependent upon their intended use, stage of drug product development, and country where the vector will be used as the different regulatory authorities who oversee the clinical usage of such products may have different requirements. As such, successful GMP manufacture of LV requires a combination of diverse factors including: regulatory expertise, compliant facilities, validated and calibrated equipments, starting materials of the highest quality, trained production personnel, scientifically robust production processes, and a quality by design approach. More importantly, oversight throughout manufacturing by an independent Quality Assurance Unit who has the authority to reject or approve the materials is required. We describe here the GMP manufacture of LV at our facility using a four plasmid system where 293T cells from an approved Master Cell Bank (MCB) are transiently transfected using polyethylenimine (PEI). Following transfection, the media is changed and Benzonase added to digest residual plasmid DNA. Two harvests of crude supernatant are collected and then clarified by filtration. The clarified supernatant is purified and concentrated by anion exchange chromatography and tangential flow filtration. The final product is then diafiltered directly into the sponsor defined final formulation buffer and aseptically filled.

Generation of High-Titer Self-Inactivated γ-Retroviral Vector Producer Cells

The γ-retroviral vector is a gene delivery vehicle that is commonly used in gene therapy. Despite its efficacy, its strong enhancers contributed to malignant transformations in some hematopoietic stem cell (HSC) gene therapy trials. A safer version without viral enhancers (SIN) is available, but its production is cumbersome, as high titers can only be obtained in transient transfection. Our aim was to develop a system that could easily generate high-titer SIN vectors from stable producer cells. The use of the cytomegalovirus enhancer-promoter sequence to generate the full-length genomic RNA combined to sequences that decrease transcriptional readthrough (WPRE and strong polyadenylation sequences) led to 6 × 106 infectious units (IU)/mL of a SIN GFP vector in transient transfection. The incorporation of a blasticidin selection cassette to the retroviral plasmid allowed the generation of stable clones in the 293Vec packaging cells that release 2 × 107 IU/mL and 1.4 × 107 IU/mL of a SIN GFP and a SIN PIGA vector, respectively. A titer of 1.8 × 106 IU/mL was obtained with a SIN vector containing the long 8.9-kb COL7A1 cDNA. Thus, an efficient process was established for the generation of stable 293Vec-derived retrovirus producer cells that release high-titer SIN vectors.

Superior lentiviral vectors designed for BSL-0 environment abolish vector mobilization

Lentiviral vector mobilization following HIV-1 infection of vector-transduced cells poses biosafety risks to vector-treated patients and their communities. The self-inactivating (SIN) vector design has reduced, however, not abolished mobilization of integrated vector genomes. Furthermore, an earlier study demonstrated the ability of the major product of reverse transcription, a circular SIN HIV-1 vector comprising a single-LTR to support production of high vector titers. Here, we demonstrate that configuring the internal vector expression cassette in opposite orientation to the LTRs abolishes mobilization of SIN vectors. This additional SIN mechanism is in part premised on induction of host PKR response to double-stranded RNAs comprised of mRNAs transcribed from cryptic transcription initiation sites around 3’SIN-LTR’s and the vector internal promoter. As anticipated, PKR response following transfection of opposite orientation vectors, negatively affects their titers. Importantly, shRNA-mediated knockdown of PKR rendered titers of SIN HIV-1 vectors comprising opposite orientation expression cassettes comparable to titers of conventional SIN vectors. High titer vectors carrying an expression cassette in opposite orientation to the LTRs efficiently delivered and maintained high levels of transgene expression in mouse livers. This study establishes opposite orientation expression cassettes as an additional PKR-dependent SIN mechanism that abolishes vector mobilization from integrated and episomal SIN lentiviral vectors.

Abstract 1541: Directed, unbiased mapping of lentiviral integrations with next generation sequencing in CAR-T cells

Abstract During manufacturing of an exemplary CD19-directed CAR-T cell product, patient T cells were transduced with a viral vector encoding the CD19-directed, 41BBζ endodomain-containing chimeric antigen receptor (CAR) construct, enabling permanent genetic modification of the target T cells. Lentiviral transduction with a self-inactivating (SIN) vector can offer certain advantages in safety and consistency over other DNA delivery methods. Although the integration patterns of wildtype HIV-1 have been well-characterized and shown to have a low risk of insertional transformation, the addition of an artificial construct may have the potential to affect integration patterns. Existing techniques for viral integration mapping, such as ligation-mediated polymerase chain reaction (LM-PCR), have limitations precluding comprehensive, unbiased, high-throughput insertion site mapping. To characterize insertional events in engineered CAR-T cells, we developed an unbiased NGS assay and computational pipeline capable of characterizing viral integration events genome-wide. The method leverages Tn5 transposase, coupled with a long terminal repeat-specific PCR, to map integration events down to nulceotide resolution. This assay identified over 150,000 high-confidence integration events across 23 patient-derived CAR-T cell products. Analysis of these sites revealed a similar integration preference to lentiviruses described in the literature: driven by GC content, local nucleotide content, chromatin status, and transcriptional activity, with no indication of preferential integration at oncogenic loci. In conclusion, in this assay a CD19-directed, CAR-containing lentivirus exhibited a low risk integration profile with no significant evidence of construct-specific risk factors. Furthermore, the findings suggest that the NGS assay may be useful for more efficient, cost-effective, and sensitive measurement of CAR-T integration events for a range of constructs. Citation Format: Timothy G. Johnstone, Rajagopal Chari, David Koppstein, Ronald J. Hause, Rafael Ponce, Adrian W. Briggs, Francois Vigneault. Directed, unbiased mapping of lentiviral integrations with next generation sequencing in CAR-T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1541.

Differences in the internalization of self-inactivating VSVG-pseudotyped murine leukemia virus-based vectors in human and murine cells

Self-inactivating VSVG-pseudotyped murine leukemia virus (SIN-VSVG-MLV) has been widely used to generate stable cell lines and produce gene delivery vectors. Despite the broad cellular tropism of the VSVG-pseudotyped MLV, we observed differential viral transduction efficiency depending on the host cell type used. In order to determine the mechanism underlying these differences, we used a GFP-expressing SIN-VSVG-MLV and analyzed the major steps of viral transduction in different cell lines including human epithelial, T-lymphocytes, monocytes and murine fibroblast cells.We observed the better transduction efficiency in HeLa cells, which was 20-fold higher than THP-1 and NIH/3T3 cells. To quantify viral internalization, we determined genomic RNA content by quantifying the early reverse transcription product. Genomic RNA and transduction levels were correlated with HeLa cells showing the higher amount of early RT product followed by tsA201 cells, while NIH/3T3, Jurkat and THP-1 had the lowest amounts. Similar results were observed when the late reverse transcription product was analyzed. Reverse transcription efficiency was 66–85% in HeLa cells and about 30% in tsA201, NIH/3T3, Jurkat and THP-1 cells. Viral integration, determined by Alu-Nested-qPCR, was higher for HeLa and lowerst for Jurkat and THP-1 cells. Interestingly, we observed that viral entry was correlated with the cellular availability of clathrin-mediated endocytosis, which was higher in HeLa and tsA201 cells, potentially explaining the higher rates of SIN-VSVG-MLV transduction and early RT synthesis observed in these cell lines.In conclusion, the SIN-VSVG-MLV vector showed significantly different rates of infectivity depending on the host cell type, possibly due to differential rates of viral internalization.

Foamy Virus Vector Carries a Strong Insulator in Its Long Terminal Repeat Which Reduces Its Genotoxic Potential

Strong viral enhancers in gammaretrovirus vectors have caused cellular proto-oncogene activation and leukemia, necessitating the use of cellular promoters in "enhancerless" self-inactivating integrating vectors. However, cellular promoters result in relatively low transgene expression, often leading to inadequate disease phenotype correction. Vectors derived from foamy virus, a nonpathogenic retrovirus, show higher preference for nongenic integrations than gammaretroviruses/lentiviruses and preferential integration near transcriptional start sites, like gammaretroviruses. We found that strong viral enhancers/promoters placed in foamy viral vectors caused extremely low immortalization of primary mouse hematopoietic stem/progenitor cells compared to analogous gammaretrovirus/lentivirus vectors carrying the same enhancers/promoters, an effect not explained solely by foamy virus' modest insertional site preference for nongenic regions compared to gammaretrovirus/lentivirus vectors. Using CRISPR/Cas9-mediated targeted insertion of analogous proviral sequences into the LMO2 gene and then measuring LMO2 expression, we demonstrate a sequence-specific effect of foamy virus, independent of insertional bias, contributing to reduced genotoxicity. We show that this effect is mediated by a 36-bp insulator located in the foamy virus long terminal repeat (LTR) that has high-affinity binding to the CCCTC-binding factor. Using our LMO2 activation assay, LMO2 expression was significantly increased when this insulator was removed from foamy virus and significantly reduced when the insulator was inserted into the lentiviral LTR. Our results elucidate a mechanism underlying the low genotoxicity of foamy virus, identify a novel insulator, and support the use of foamy virus as a vector for gene therapy, especially when strong enhancers/promoters are required.IMPORTANCE Understanding the genotoxic potential of viral vectors is important in designing safe and efficacious vectors for gene therapy. Self-inactivating vectors devoid of viral long-terminal-repeat enhancers have proven safe; however, transgene expression from cellular promoters is often insufficient for full phenotypic correction. Foamy virus is an attractive vector for gene therapy. We found foamy virus vectors to be remarkably less genotoxic, well below what was expected from their integration site preferences. We demonstrate that the foamy virus long terminal repeats contain an insulator element that binds CCCTC-binding factor and reduces its insertional genotoxicity. Our study elucidates a mechanism behind the low genotoxic potential of foamy virus, identifies a unique insulator, and supports the use of foamy virus as a vector for gene therapy.

British Society for Gene and Cell Therapy Annual Conference and Joint UK Regenerative Medicine Platform Meeting Royal Welsh College of Music & Drama Cardiff, Wales, United Kingdom Wednesday April 19-Friday April 21, 2017 Conference Abstracts.

British Society for Gene and Cell Therapy Annual Conference and Joint UK Regenerative Medicine Platform Meeting Royal Welsh College of Music & Drama Cardiff, Wales, United Kingdom Wednesday April 19-Friday April 21, 2017 Conference Abstracts.

The retroviral vector family: something for everyone.

After 30years of retroviral vector research it became clear that the parental viruses can be both friend and foe. Especially human immunodeficiency virus sparked a global pandemic, but could be converted into a versatile tool for cell therapy. For all retroviral genera, the way from virus to vector was similar resulting in split-vector systems based on the separation of the genes needed for vector particle formation and transgene expression. The first gene therapy trials, although clinically effective, revealed the genotoxicity of retroviral vectors caused by insertional mutagenesis. This issue was solved using self-inactivating vectors carrying weaker cellular promoters. Further fine-tuning was able to generate inducible systems. The current toolbox also contains vectors for the generation of induced pluripotent stem cells or efficient RNA interference. More recently the application of CRISPR-Cas9-mediated gene editing led to the development of genome-wide small guide RNA libraries targeting all human genes and single lentiviral vectors for an easy delivery of Cas9.

Alpharetroviral self-inactivating vectors: an emerging tool for gene therapy

Alpharetroviral self-inactivating vectors: an emerging tool for gene therapy

Viral Vectors: The Road to Reducing Genotoxicity.

Viral vector use in gene therapy has highlighted several safety concerns, including genotoxic events. Generally, vector-mediated genotoxicity results from upregulation of cellular proto-oncogenes via promoter insertion, promoter activation, or gene transcript truncation, with enhancer-mediated activation of nearby genes the primary mechanism reported in gene therapy trials. Vector-mediated genotoxicity can be influenced by virus type, integration target site, and target cell type; different vectors have distinct integration profiles which are cell-specific. Non-viral factors, including patient age, disease, and dose can also influence genotoxic potential, thus the choice of test models and clinical trial populations is important to ensure they are indicative of efficacy and safety. Efforts have been made to develop viral vectors with less risk of insertional mutagenesis, including self-inactivating (SIN) vectors, enhancer-blocking insulators, and microRNA targeting of vectors, although insertional mutagenesis is not completely abrogated. Here we provide an overview of the current understanding of viral vector-mediated genotoxicity risk from factors contributing to viral vector-mediated genotoxicity to efforts made to reduce genotoxicity, and testing strategies required to adequately assess the risk of insertional mutagenesis. It is clear that there is not a 'one size fits all' approach to vector modification for reducing genotoxicity, and addressing these challenges will be a key step in the development of therapies such as CRISPR-Cas9 and delivery of future gene-editing technologies.

Lentivirus Mediated Correction of Artemis-Deficient Severe Combined Immunodeficiency.

During B and T lymphocyte maturation, V(D)J recombination is initiated by creation of DNA double-strand breaks. Artemis is an exonuclease essential for their subsequent repair by nonhomologous end-joining. Mutations in DCLRE1C, the gene encoding Artemis, cause T-B-NK+ severe combined immunodeficiency (ART-SCID) and also confer heightened sensitivity to ionizing radiation and alkylating chemotherapy. Although allogeneic hematopoietic cell transplantation can treat ART-SCID, conditioning regimens are poorly tolerated, leading to early mortality and/or late complications, including short stature, endocrinopathies, and dental aplasia. However, without alkylating chemotherapy as preconditioning, patients usually have graft rejection or limited T cell and no B cell recovery. Thus, addition of normal DCLRE1C cDNA to autologous hematopoietic stem cells is an attractive strategy to treat ART-SCID. We designed a self-inactivating lentivirus vector containing human Artemis cDNA under transcriptional regulation of the human endogenous Artemis promoter (AProArt). Fibroblasts from ART-SCID patients transduced with AProArt lentivirus showed correction of radiosensitivity. Mobilized peripheral blood CD34+ cells from an ART-SCID patient as well as hematopoietic stem cells from Artemis-deficient mice demonstrated restored T and B cell development following AProArt transduction. Murine hematopoietic cells transduced with AProArt exhibited no increase in replating potential in an in vitro immortalization assay, and analysis of AProArt lentivirus insertions showed no predilection for sites that could activate oncogenes. These efficacy and safety findings support institution of a clinical trial of gene addition therapy for ART-SCID.

Rapid and Efficient Stable Gene Transfer to Mesenchymal Stromal Cells Using a Modified Foamy Virus Vector.

Mesenchymal stromal cells (MSCs) hold great promise for regenerative medicine. Stable ex vivo gene transfer to MSCs could improve the outcome and scope of MSC therapy, but current vectors require multiple rounds of transduction, involve genotoxic viral promoters and/or the addition of cytotoxic cationic polymers in order to achieve efficient transduction. We describe a self-inactivating foamy virus vector (FVV), incorporating the simian macaque foamy virus envelope and using physiological promoters, which efficiently transduces murine MSCs (mMSCs) in a single-round. High and sustained expression of the transgene, whether GFP or the lysosomal enzyme, arylsulphatase A (ARSA), was achieved. Defining MSC characteristics (surface marker expression and differentiation potential), as well as long-term engraftment and distribution in the murine brain following intracerebroventricular delivery, are unaffected by FVV transduction. Similarly, greater than 95% of human MSCs (hMSCs) were stably transduced using the same vector, facilitating human application. This work describes the best stable gene transfer vector available for mMSCs and hMSCs.

Alpharetroviral self-inactivating vectors produced by a superinfection-resistant stable packaging cell line allow genetic modification of primary human T lymphocytes

Primary human T lymphocytes represent an important cell population for adoptive immunotherapies, including chimeric-antigen and T-cell receptor applications, as they have the capability to eliminate non-self, virus-infected and tumor cells. Given the increasing numbers of clinical immunotherapy applications, the development of an optimal vector platform for genetic T lymphocyte engineering, which allows cost-effective high-quality vector productions, remains a critical goal. Alpharetroviral self-inactivating vectors (ARV) have several advantages compared to other vector platforms, including a more random genomic integration pattern and reduced likelihood for inducing aberrant splicing of integrated proviruses. We developed an ARV platform for the transduction of primary human T lymphocytes. We demonstrated functional transgene transfer using the clinically relevant herpes-simplex-virus thymidine kinase variant TK.007. Proof-of-concept of alpharetroviral-mediated T-lymphocyte engineering was shown in vitro and in a humanized transplantation model in vivo. Furthermore, we established a stable, human alpharetroviral packaging cell line in which we deleted the entry receptor (SLC1A5) for RD114/TR-pseudotyped ARVs to prevent superinfection and enhance genomic integrity of the packaging cell line and viral particles. We showed that superinfection can be entirely prevented, while maintaining high recombinant virus titers. Taken together, this resulted in an improved production platform representing an economic strategy for translating the promising features of ARVs for therapeutic T-lymphocyte engineering.