Vector Insertion Research Articles

Metabolic engineering of Rhodococcus jostii RHA1 for production of pyridine-dicarboxylic acids from lignin

Genetic modification of Rhodococcus jostii RHA1 was carried out in order to optimise the production of pyridine-2,4-dicarboxylic acid and pyridine-2,5-dicarboxylic acid bioproducts from lignin or lignocellulose breakdown, via insertion of either the Sphingobium SYK-6 ligAB genes or Paenibacillus praA gene respectively. Insertion of inducible plasmid pTipQC2 expression vector containing either ligAB or praA genes into a ΔpcaHG R. jostii RHA1 gene deletion strain gave 2–threefold higher titres of PDCA production from lignocellulose (200–287 mg/L), compared to plasmid expression in wild-type R. jostii RHA1. The ligAB genes were inserted in place of the chromosomal pcaHG genes encoding protocatechuate 3,4-dioxygenase, under the control of inducible Picl or PnitA promoters, or a constitutive Ptpc5 promoter, producing 2,4-PDCA products using either wheat straw lignocellulose or commercial soda lignin as carbon source. Insertion of Amycolatopsis sp. 75iv2 dyp2 gene on a pTipQC2 expression plasmid led to enhanced titres of 2,4-PDCA products, due to enhanced rate of lignin degradation. Growth in minimal media containing wheat straw lignocellulose led to the production of 2,4-PDCA in 330 mg/L titre in 40 h, with > tenfold enhanced productivity, compared with plasmid-based expression of ligAB genes in wild-type R. jostii RHA1. Production of 2,4-PDCA was also observed using several different polymeric lignins as carbon sources, and a titre of 240 mg/L was observed using a commercially available soda lignin as feedstock.

Evaluation of Node-Metastasis in Sparse Underwater Acoustic Sensor Networks for Localization Under Acoustically Stratified Malicious Node Conditions

Malicious anchor node is a serious issue in underwater sensor networks. This problem gets compounded under sparse deployment scenario. To overcome this issue, the authors present a compressed sensing theorem (abbreviated as CS-MC Theorem) which would be applicable to underwater localization. To counter malicious node behavior, CS-MC Theorem is justified through three lemmas. For underwater acoustic propagation, a specific L1-norm minimization method is derived. It describes the condition for unique localization of target by discretizing the target search space. Malicious node is identified by inserting the anchor nodes into the discretized grid. An insertion vector concept is introduced to determine the extent of node metastasis. Derivations are performed to prove the mathematical basis of the proposed algorithm under acoustically stratified and unstratified scenarios. Numerical results establish the feasibility of CS-MC algorithm for sparse underwater acoustic sensor network.

Efficient marking of human cells with rapid but transient repopulating activity in autografted recipients

AbstractShort-term hematopoietic reconstituting cells have been identified in mice, nonhuman primates, and among human cells that engraft xenogeneic hosts. We now present clonal marking data demonstrating a rapid but unsustained contribution of cultured human autografts to the initial phase of hematologic recovery in myeloablated patients. Three patients received transplants of granulocyte colony-stimulating factor–mobilized autologous peripheral blood (P444448) cells, of which a portion (8%-25% of the CD34+ cells) had been incubated in vitro with growth factors (5 days) and clinical grade LN retrovirus (3-5 days). More than 9% of the clonogenic and long-term culture-initiating cells harvested were transduced. Semiquantitative and linear amplification-mediated polymerase chain reaction analyses of serial PB samples showed that marked white blood cells appeared in all 3 patients within 11 days and transiently constituted up to 0.1% to 1% of those produced in the first month. However, within another 2 to 9 months, marked cells had permanently decreased to very low levels. Analysis of more than 50 vector insertion sites showed none of the clones detected in the first month were active later. Eighty percent of inserts were located within or near genes, 2 near CXCR4. These findings provide direct evidence of cells with rapid but transient repopulating activity in patients and demonstrate their efficient transduction in vitro.

Multiple Genes Surrounding Bcl-xL, a Common Retroviral Insertion Site, Can Influence Hematopoiesis Individually or in Concert

Retroviral insertional mutagenesis (RIM) is both a relevant risk in gene therapy and a powerful tool for identifying genes that enhance the competitiveness of repopulating hematopoietic stem and progenitor cells (HSPCs). However, focusing only on the gene closest to the retroviral vector insertion site (RVIS) may underestimate the effects of RIM, as dysregulation of distal and/or multiple genes by a single insertion event was reported in several studies. As a proof of concept, we examined the common insertion site (CIS) Bcl-xL, which revealed seven genes located within ±150 kb from the RVIS for our study. We confirmed that Bcl-xL enhanced the competitiveness of HSPCs, whereas the Bcl-xL neighbor Id1 hindered HSPC long-term repopulation. This negative influence of Id1 could be counteracted by co-expressing Bcl-xL. Interestingly, >90% of early reconstituted myeloid cells were found to originate from transduced HSPCs upon simultaneous overexpression of Bcl-xL and Id1, which implies that Bcl-xL and Id1 can collaborate to rapidly replenish the myeloid compartment under stress conditions. To directly compare the competitiveness of HSPCs conveyed by multiple transgenes, we developed a multiple competitor competitive repopulation (MCCR) assay to simultaneously screen effects on HSPC repopulating capacity in a single mouse. The MCCR assay revealed that multiple genes within a CIS can have positive or negative impact on hematopoiesis. Furthermore, these data highlight the importance of studying multiple genes located within the proximity of an insertion site to understand complex biological effects, especially as the number of gene therapy patients increases.

A Micro-Computed Tomography Study of Round Window Anatomy and Implications for Atraumatic Cochlear Implant Insertion.

The goal of this study was to interrogate high-resolution three-dimensional reconstructions of round window anatomy to illustrate and characterize structural variability with implications for atraumatic cochlear implant insertion. Cochlear implants are increasingly used to improve sound detection in patients with substantial residual hearing. However, traumatic cochlear implant insertion through the round window involving upward deviation of the electrode into the spiral ligament, basilar membrane, and osseous spiral lamina, medial impaction on the modiolus, or interscalar excursion into the scala vestibuli are associated with lower rates of hearing preservation and poorer speech perception.Successful atraumatic insertion is dependent on an anatomical understanding of the middle and inner ear. The round window bony niche lacks distinct demonstrable anatomical landmarks for the position of the round window membrane, and there is limited guidance on the amount of bony overhang that can be safely drilled away. A greater understanding of the anatomical variation around the round window could enhance treatment efficacy. Fourteen human cadaver temporal bones were imaged using microcomputed tomography. Resulting scans were digitally reconstructed, segmented, and measured. Round window niche walls vary substantially in size and projection. Round window average short diameter measured 1.30 mm (range 1.07-1.44), and is limited by the crista fenestrae at the inferoanterior margin of the round window. Crista fenestrae size and morphology varied considerably. Reconstructions with solid and translucent panels are presented. Anatomical heterogeneity should be considered in cochlear implant selection, drilling, and choice of insertion vector.

Recombinant Fowlpox Virus Expressing gB Gene from Predominantly Epidemic Infectious Larygnotracheitis Virus Strain Demonstrates Better Immune Protection in SPF Chickens.

Background: Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens. Antigenic mutation of infectious laryngotracheitis virus (ILTV) may result in a vaccination failure in the poultry industry and thus a protective vaccine against predominant ILTV strains is highly desirable. Methods: The full-length glycoprotein B (gB) gene of ILTV with the two mutated synonymous sites of fowlpox virus (FPV) transcription termination signal sequence was cloned into the insertion vector p12LS, which was co-transfected with wild-type (wt) FPV into chicken embryo fibroblast (CEF) to develop a recombinant fowlpox virus-gB (rFPV-gB) candidate vaccine strain. Furthermore, its biological and immunological characteristics were evaluated. Results: The results indicated that gB gene was expressed correctly in the rFPV by indirect immunofluorescent assay and Western blot, and the rFPV-gB provided a 100% protection in immunized chickens against the challenge of predominant ILTV strains that were screened by pathogenicity assay when compared with the commercialized rFPV vaccine, which only provided 83.3%. Conclusion: rFPV-gB can be used as a potential vaccine against predominant ILTV strains.

Hematopoietic Tumors in a Mouse Model of X-linked Chronic Granulomatous Disease after Lentiviral Vector-Mediated Gene Therapy

Chronic granulomatous disease (CGD) is a rare inherited disorder due to loss-of-function mutations in genes encoding the NADPH oxidase subunits. Hematopoietic stem and progenitor cell (HSPC) gene therapy (GT) using regulated lentiviral vectors (LVs) has emerged as a promising therapeutic option for CGD patients. We performed non-clinical Good Laboratory Practice (GLP) and laboratory-grade studies to assess the safety and genotoxicity of LV targeting myeloid-specific Gp91phox expression in X-linked chronic granulomatous disease (XCGD) mice. We found persistence of gene-corrected cells for up to 1 year, restoration of Gp91phox expression and NADPH oxidase activity in XCGD phagocytes, and reduced tissue inflammation after LV-mediated HSPC GT. Although most of the mice showed no hematological or biochemical toxicity, a small subset of XCGD GT mice developed T cell lymphoblastic lymphoma (2.94%) and myeloid leukemia (5.88%). No hematological malignancies were identified in C57BL/6 mice transplanted with transduced XCGD HSPCs. Integration pattern analysis revealed an oligoclonal composition with rare dominant clones harboring vector insertions near oncogenes in mice with tumors. Collectively, our data support the long-term efficacy of LV-mediated HSPC GT in XCGD mice and provide a safety warning because the chronic inflammatory XCGD background may contribute to oncogenesis.

Insertion Depth for Optimized Positioning of Precurved Cochlear Implant Electrodes.

Generic guidelines for insertion depth of precurved electrodes are suboptimal for many individuals. Insertion depths that are too shallow result in decreased cochlear coverage, and ones that are too deep lift electrodes away from the modiolus and degrade the electro-neural interface. Guidelines for insertion depth are generically applied to all individuals using insertion depth markers on the array that can be referenced against anatomical landmarks. To normalize our measurements, we determined the optimal position and insertion vector where a precurved array best fits the cochlea for each patient in an IRB-approved, N = 131 subject CT database. The distances from the most basal electrode on an optimally placed array to anatomical landmarks, including the round window (RW) and facial recess (FR), was measured for all patients. The standard deviations of the distance from the most basal electrode to the FR and RW are 0.65 mm and 0.26 mm, respectively. Owing to the high variability in FR distance, using the FR as a landmark to determine insertion depth results in >0.5 mm difference with ideal depth in 44% of cases. Alignment of either of the two most proximal RW markers with the RW would result in over-insertion failures for >80% of cases, whereas the use of the third, most medial marker would result in under-insertion in only 19% of cases. Normalized measurements using the optimized insertion vector show low variance in distance from the basal electrode position to the RW, thereby suggesting it as a better landmark for determining insertion depth than the FR.

Lentiviral Hematopoietic Stem Cell Gene Therapy Corrects Murine Pompe Disease

Pompe disease is an autosomal recessive lysosomal storage disorder characterized by progressive muscle weakness. The disease is caused by mutations in the acid α-glucosidase (GAA) gene. Despite the currently available enzyme replacement therapy (ERT), roughly half of the infants with Pompe disease die before the age of 3 years. Limitations of ERT are immune responses to the recombinant enzyme, incomplete correction of the disease phenotype, lifelong administration, and inability of the enzyme to cross the blood-brain barrier. We previously reported normalization of glycogen in heart tissue and partial correction of the skeletal muscle phenotype by ex vivo hematopoietic stem cell gene therapy. In the present study, using a codon-optimized GAA (GAAco), the enzyme levels resulted in close to normalization of glycogen in heart, muscles, and brain, and in complete normalization of motor function. A large proportion of microglia in the brain was shown to be GAA positive. All astrocytes contained the enzyme, which is in line with mannose-6-phosphate receptor expression and the key role in glycogen storage and glucose metabolism. The lentiviral vector insertion site analysis confirmed no preference for integration near proto-oncogenes. This correction of murine Pompe disease warrants further development toward a cure of the human condition.

Genome-Wide profiling of viral vector integration in clinical CAR T products using comprehensive methodologies

Background & Aim Chimeric antigen receptor (CAR)-engineered T cells have become an important cancer immunotherapy and the clinical application of these cells is increasing rapidly. For clinical CAR T-cell therapies, autologous T-cells are genetically modified to express a specific/bispecific chimeric antigen receptor. These CAR T-cells have high specificity and affinity for antigens expressed on the surface of target cells. At present, T-cells are genetically modified by introduction of the receptor by gamma- and lentiviral vectors, which integrate into the cell genome and is subsequently expressed. However, as transduction efficiency and the number of integration events increases, the risk of insertional mutagenesis also increases. In order to maintain the safety and effectiveness of clinical CAR T-cells, it is essential to monitor the insertion efficiency, accurately assess insertion sites and determine the effects of vector instertion on the CAR T-cell transcriptome profile. Methods, Results & Conclusion We developed a comprehensive methodology for evaluating the safety and effectiveness of CAR T-cells, utilizing droplet digital PCR to measure vector copy number, DNA-seq to measure vector insertion sites and RNA-seq to assess gene expression profiles. These assays were applied to 38 clinical CAR T products (18 gamma- and 20 lentiviral vector). We verified that ddPCR can reliably measure the average number of vector copies integrated into the CAR T-cell genome. CAR T products with a detectable number of vector copies were subjected to DNA-seq. The results showed that DNA-seq accurately revealed integration site maps in the whole genome and found some loci that were integration hot spots. Moreover, we found gamma-retroviral vectors resulted in more integration events than lentiviral vectors. Combining the RNA-seq and DNA-seq data revealed that most integration events within coding genes did not result in a change in gene expression. We did noticed that some gene loci both have an integration event and changed gene expression. Further studies will focus on these finding and the impact on CAR T-cell clinical outcomes. In summary, we established a series of methods to detect vector copy number, insertion sites and gene expression profiles in clincial CAR T products. These methods will be useful for exploring the risk of insertional mutagenesis of viral vector in CAR T products, the signficance of specific integration sites, and facilates improved safety in development of CAR T cell therapies.

TEG001 insert integrity from vector producer cells until medicinal product

Background & Aim Despite extensive usage of gene therapy medicinal products (GTMPs) in clinical studies and recent approval of CAR T cell therapy, little information has been made available on the precise molecular characterization and possible variations in terms of insert integrity and vector copy numbers of different GTMPs during the complete production chain. Within this context we characterize αβT cells Engineered to express a defined γδT cell receptor (TEGs) currently used in a first-in-human clinical study (NTR6541). Methods, Results & Conclusion Utilizing Targeted Locus Amplification in combination with Next Generation Sequencing for the vector producer clone and TEG001 products we report on 5 single nucleotide variants and 9 intact vector copies integrated in the producer clone. The vector copy number in TEG001 cells was on average a factor 0.72 (SD 0.11) below that of the producer cell clone. All nucleotide variants were transferred to TEG001, without having an effect on cellular proliferation during extensive in vitro culture. Based on an environmental risk assessment of the 5 nucleotide variants present in the non-coding viral region of the TEG001 insert, there was no altered environmental impact of TEG001 cells. We conclude that TEG001 cells do not have an increased risk for malignant transformation in vivo. In conclusion, we have reported an extensive molecular characterization of TEG001 transgene integrity that resulted in the approval a phase I clinical study which did not only allow to investigate the safety and tolerability of TEG001 in patients with relapsed/refractory acute myeloid leukemia, high-risk myelodysplastic syndrome, and relapsed/refractory multiple myeloma but also will provide a valuable framework for the molecular characterisation of future GTMPs.

Manufacturing Development of SENTI-101, a Gene Circuit Modified Allogeneic Bone Marrow Derived Mesenchymal Stromal Cell (BM-MSC) Therapy for the Treatment of Solid Tumors

Background & Aim Immunotherapies have shown success in activating endogenous immune responses against solid tumors. Many immunologically cold tumors do not respond to current immunotherapies such as checkpoint inhibitors, and present a major unmet clinical need. In prior clinical studies, allogeneic gene modified MSCs have been administered through intraperitoneal (IP) infusion to ovarian cancer patients, demonstrating safety and localization to tumor lesions.1 SENTI-101 is an allogeneic cell product consisting of MSCs engineered with a gene-circuit that expresses a potent combination of immunomodulatory cytokines: IL12 and IL21. Upon IP administration, SENTI-101 innately homes to peritoneal tumors, secretes IL12 and IL21 in a localized and sustained fashion, and induces a strong and durable anti-tumor immune response. Methods, Results & Conclusion The gene modification process for SENTI-101 includes packaging the gene circuit into a lentiviral vector, stable transduction of allogeneic BM-MSCs, expansion, and cryopreservation. The drug product is further expanded, formulated, and cryopreserved prior to clinical delivery. Key product attributes of SENTI-101 include: transduction efficiency, IL12 and IL21 secretion, cell doubling time, tumor homing, and vector copy number. A 2D culture manufacturing process was developed to maintain key product attributes over at least 14 population doublings, translating to over 10^12 final product cells per bone marrow collection. The product attributes were comparable between freshly cultured cells and those that underwent a freeze/thaw cycle and further expanded. They also showed similar localized cytokine production of IL12 and IL21 and in vivo efficacy in tumor models. Transduction efficiency was optimized by comparing different MSC donors, lentivirus titer, different chemical transduction enhancers, and other process variables. SENTI-101 was further evaluated for growth characteristics, immunomodulatory function, distribution and kinetics in mouse models, vector insertion site analysis, cell transformation assay, and karyotype. In addition, SENTI-101 was demonstrated to maintain equivalent product attributes in a 3D bioreactor process, enabling larger scale production for the allogeneic MSCs for commercial manufacturing. In conclusion, we have developed a GMP-ready process for the gene modification, expansion, and administration of SENTI-101 in preparation for upcoming human clinical studies in solid tumor indications.

Magnetically Steered Robotic Insertion of Cochlear-Implant Electrode Arrays: System Integration and First-In-Cadaver Results.

Cochlear-implant electrode arrays (EAs) must be inserted accurately and precisely to avoid damaging the delicate anatomical structures of the inner ear. It has previously been shown on the benchtop that using magnetic fields to steer magnet-tipped EAs during insertion reduces insertion forces, which correlate with insertion errors and damage to internal cochlear structures. This paper presents several advancements toward the goal of deploying magnetic steering of cochlear-implant EAs in the operating room. In particular, we integrate image guidance with patient-specific insertion vectors, we incorporate a new nonmagnetic insertion tool, and we use an electromagnetic source, which provides programmable control over the generated field. The electromagnet is safer than prior permanent-magnet approaches in two ways: it eliminates motion of the field source relative to the patient's head and creates a field-free source in the power-off state. Using this system, we demonstrate system feasibility by magnetically steering EAs into a cadaver cochlea for the first time. We show that magnetic steering decreases average insertion forces, in comparison to manual insertions and to image-guided robotic insertions alone.

Human Cochlear Nucleus on 7 Tesla Diffusion Tensor Imaging: Insights Into Micro-anatomy and Function for Auditory Brainstem Implant Surgery.

The cochlear nucleus (CN) is the target of the auditory brainstem implant (ABI). Most ABI candidates have Neurofibromatosis Type 2 (NF2) and distorted brainstem anatomy from bilateral vestibular schwannomas. The CN is difficult to characterize as routine structural MRI does not resolve detailed anatomy. We hypothesize that diffusion tensor imaging (DTI) enables both in vivo localization and quantitative measurements of CN morphology. We analyzed 7 Tesla (T) DTI images of 100 subjects (200 CN) and relevant anatomic structures using an MRI brainstem atlas with submillimetric (50 μm) resolution. Tertiary referral center. Young healthy normal hearing adults. Diagnostic. Diffusion scalar measures such as fractional anisotropy (FA), mean diffusivity (MD), mode of anisotropy (Mode), principal eigenvectors of the CN, and the adjacent inferior cerebellar peduncle (ICP). The CN had a lamellar structure and ventral-dorsal fiber orientation and could be localized lateral to the inferior cerebellar peduncle (ICP). This fiber orientation was orthogonal to tracts of the adjacent ICP where the fibers run mainly caudal-rostrally. The CN had lower FA compared to the medial aspect of the ICP (0.44 ± 0.09 vs. 0.64 ± 0.08, p < 0.001). 7T DTI enables characterization of human CN morphology and neuronal substructure. An ABI array insertion vector directed more caudally would better correspond to the main fiber axis of CN. State-of-the-art DTI has implications for ABI preoperative planning and future image guidance-assisted placement of the electrode array.

Conditional Immortalization of Lymphoid Progenitors via Tetracycline-Regulated LMO2 Expression.

Conditional immortalization of hematopoietic progenitors through lentiviral expression of selected transcription factors in hematopoietic stem and progenitor cells provides a promising tool to study stem cell and leukemia biology. In this study, to generate conditionally immortalized lymphoid progenitor (ciLP) cell lines, murine hematopoietic progenitor cells were transduced with an inducible lentiviral "all-in-one" vector expressing LMO2 under doxycycline (DOX) stimulation and the reverse tetracycline-regulated transactivator (rtTA3). For selection of LMO2-expressing ciLPs (LMO2-ciLPs) and longitudinal manipulation in T cell differentiation lymphoid conditions, we developed a robust approach based on coculture with OP9-DL1 stromal cells and improved cytokine conditions allowing a controlled balance between cell proliferation and differentiation in vitro. LMO2-ciLP cell lines with the highest proliferation, vector copy number, and similar insertion pattern were selected for LMO2 "on/off" in vitro study. LMO2 expression under DOX induction resulted in a double negative (DN) 2 differentiation arrest and a propagation of CD44+CD25- myeloid cell population characterized by lymphoid and myeloid phenotypes, respectively. Both DN2 and CD44+CD25- myeloid cell subpopulations expressed c-KIT, suggesting that LMO2-ciLPs were similar to uncommitted progenitors under DOX supplementation. DOX removal resulted in cessation of ectopic LMO2 expression and LMO2-ciLPs continued T cell lymphoid differentiation accompanied by c-KIT downregulation and interleukin 7 receptor expression. Switching off LMO2 expression was accompanied by increased Notch signaling and significant reduction of the CD44+CD25- myeloid cell population under T cell differentiation lymphoid conditions. Although vector insertions in cooperation with LMO2 expression could influence the fate of LMO2-ciLPs and additional experiments are required to evaluate it, our approach provides a promising tool to investigate mechanisms underlying stem cell, leukemia, and lymphocyte biology, leading to novel approaches for disease modeling and therapy evaluation.

Sphere-induced reprogramming of RPE cells into dual-potential RPE stem-like cells.

BackgroundThe retinal pigment epithelium (RPE) has the potential to regenerate the entire neuroretina upon retinal injury in amphibians. In contrast, this regenerative capacity has been lost in mammals. The reprogramming of differentiated somatic cells into induced pluripotent stem cells (iPSCs) by viral transduction of exogenous stem cell factors has triggered a revolution in regenerative medicine. However, the risks of potential mutation(s) caused by random viral vector insertion in host genomes and tumor formation in recipients hamper its clinical application. One alternative is to immortalize adult stem cells with limited potential or to partially reprogram differentiated somatic cells into progenitor-like cells through non-integration protocols.MethodsSphere-induced RPE stem cells (iRPESCs) were generated from adult mouse RPE cells. Their stem cell functionality was studied in a mouse model of retinal degeneration. The molecular mechanism underlying the sphere-induced reprogramming was investigated using microarray and loss-of-function approaches.FindingsWe provide evidence that our sphere-induced reprogramming protocol can immortalize and transform mouse RPE cells into iRPESCs with dual potential to differentiate into cells that express either RPE or photoreceptor markers both in vitro and in vivo. When subretinally transplanted into mice with retinal degeneration, iRPESCs can integrate to the RPE and neuroretina, thereby delaying retinal degeneration in the model animals. Our molecular analyses indicate that the Hippo signaling pathway is important in iRPESC reprogramming.InterpretationThe Hippo factor Yap1 is activated in the nuclei of cells at the borders of spheres. The factors Zeb1 and P300 downstream of the Hippo pathway are shown to bind to the promoters of the stemness genes Oct4, Klf4 and Sox2, thereby likely transactivate them to reprogram RPE cells into iRPESCs.FundNational Natural Science Foundation of China and the National Institute of Health USA.

TEG001 Insert Integrity from Vector Producer Cells until Medicinal Product.

Despite extensive usage of gene therapy medicinal products (GTMPs) in clinical studies and recent approval of chimeric antigen receptor (CAR) Tcell therapy, little information has been made available on the precise molecular characterization and possible variations in terms of insert integrity and vector copy numbers of different GTMPs during the complete production chain. Within this context, we characterize αβT cells engineered to express a defined γδT cell engineered to express a defined γδT receptor (TEG) currently used in a first-in-human clinical study (NTR6541). Utilizing targeted locus amplification in combination with next generation sequencing for the vector producer clone and TEG001 products, we report on five single-nucleotide variants and nine intact vector copies integrated in the producer clone. The vector copy number in TEG001 cells was on average a factor 0.72 (SD 0.11) below that of the producer cell clone. All nucleotide variants were transferred to TEG001 without having an effect on cellular proliferation during extensive invitro culture. Based on an environmental risk assessment of the five nucleotide variants present in the non-coding viral region of the TEG001 insert, there was no altered environmental impact of TEG001 cells. We conclude that TEG001 cells do not have an increased risk for malignant transformation invivo.

Oncolytic adenovirus drives specific immune response generated by a poly-epitope pDNA vaccine encoding melanoma neoantigens into the tumor site

BackgroundDNA vaccines against cancer held great promises due to the generation of a specific and long-lasting immune response. However, when used as a single therapy, they are not able to drive the generated immune response into the tumor, because of the immunosuppressive microenvironment, thus limiting their use in humans. To enhance DNA vaccine efficacy, we combined a new poly-epitope DNA vaccine encoding melanoma tumor associated antigens and B16F1-specific neoantigens with an oncolytic virus administered intratumorally.MethodsGenomic analysis were performed to find specific mutations in B16F1 melanoma cells. The antigen gene sequences were designed according to these mutations prior to the insertion in the plasmid vector. Mice were injected with B16F1 tumor cells (n = 7–9) and therapeutically vaccinated 2, 9 and 16 days after the tumor injection. The virus was administered intratumorally at day 10, 12 and 14. Immune cell infiltration analysis and cytokine production were performed by flow cytometry, PCR and ELISPOT in the tumor site and in the spleen of animals, 17 days after the tumor injection.ResultsThe combination of DNA vaccine and oncolytic virus significantly increased the immune activity into the tumor. In particular, the local intratumoral viral therapy increased the NK infiltration, thus increasing the production of different cytokines, chemokines and enzymes involved in the adaptive immune system recruitment and cytotoxic activity. On the other side, the DNA vaccine generated antigen-specific T cells in the spleen, which migrated into the tumor when recalled by the local viral therapy. The complementarity between these strategies explains the dramatic tumor regression observed only in the combination group compared to all the other control groups.ConclusionsThis study explores the immunological mechanism of the combination between an oncolytic adenovirus and a DNA vaccine against melanoma. It demonstrates that the use of a rational combination therapy involving DNA vaccination could overcome its poor immunogenicity. In this way, it will be possible to exploit the great potential of DNA vaccination, thus allowing a larger use in the clinic.

Divergent memory responses driven by adenoviral vectors are impacted by epitope competition.

Adenoviral vectors induce robust epitope‐specific CD8+ T cell responses. Within the repertoire of responses generated both conventional memory evolution and the phenomenon of memory inflation are seen. The rules governing which epitopes inflate are not fully known, but may include a role for both antigen processing and competition. To investigate this, we looked at memory generated from vectors targeting the Gp33‐41 (KAVYNFATC/K9C) epitope from the gp of lymphocytic choriomeningitis virus (LCMV) in mice. This well‐described epitope has both the Gp33‐41 and Gp34‐41 epitopes embedded within it. Vaccination with a full‐length gp or a minigene Ad‐Gp33/K9C vector‐induced conventional memory responses against the immunodominant Gp33/K9C epitope but a strong inflationary response against the Gp34/A8C epitope. These responses showed sustained in vivo function, with complete protection against LCMV infectious challenge. Given the unexpected competition between epitopes seen in the minigene model, we further tested epitope competition using the full‐length Ad‐LacZ (β‐galactosidase) model. Generation of an Ad‐LacZ vector with a single amino acid disruption of the inflationary β‐gal96‐103/D8V epitope transformed the β‐gal497‐504/I8V epitope from conventional to inflationary memory. This work collectively demonstrates the importance of epitope competition within adenoviral vector inserts and is of relevance to future studies using adenoviral vectored immunogens.

Safety and Immunogenicity of the Heterosubtypic Influenza A Vaccine MVA-NP+M1 Manufactured on the AGE1.CR.pIX Avian Cell Line.

Seasonal influenza infections have a significant global impact leading to increased health and economic burden. The efficacy of currently available seasonal influenza vaccines targeting polymorphic surface antigens has historically been suboptimal. Cellular immune responses against highly conserved Influenza A virus antigens, such as nucleoprotein (NP) and matrix protein-1 (M1), have previously been shown to be associated with protection from disease, whilst viral-vectored vaccines are an effective strategy to boost cell-mediated immunity. We have previously demonstrated that MVA encoding NP and M1 can induce potent and persistent T cell responses against influenza. In this Phase I study, we evaluated the safety and immunogenicity of MVA-NP+M1, which was newly manufactured on an immortalized cell line, in six healthy adult participants. The vaccine was well-tolerated with only mild to moderate adverse events that resolved spontaneously and were comparable to previous studies with the same vaccine manufactured in chick embryo fibroblasts. A significant increase in vaccine-specific T cell responses was detected seven days after immunization and was directed against both antigens in the vector insert. This small Phase I study supports progression of this vaccine to a Phase IIb study to assess immunogenicity and additional protective efficacy in older adults receiving licensed seasonal influenza vaccines.