Vehicles For Gene Therapy Research Articles

608. In Silico Reconstructed Ancestral Adeno-Associated Viruses Transduce Mouse Anterior Segment

Adeno-associated virus (AAV) has been widely used as a gene delivery vehicle in ocular gene therapy, especially for the treatment of inherited retinal diseases. It has been proven in animal models and humans that many AAVs can efficiently transduce different cell types in the retina. However, there are very few reports showing that single strand AAVs can successfully transduce the anterior segment of the eye. Some self-complementary AAVs, such as self- complementary AAV2 (scAAV2), have been shown to have robust transduction in the anterior segment cells of mouse, rat, and monkey. Unfortunately, the maximum package genome size of the self-complementary AAV is approximately 2.2kbp, which is less than half the package capacity of single strand AAV vectors. In previous studies, we have shown that an ancestral AAV, Anc80, which was developed in our lab by in silico ancestral sequence reconstruction, can achieve fast onset and long-term stable expression in retinal cells. In the present study, we have explored the tropism and transduction efficiency of the ancestral AAV vector (Anc80) in the anterior segment of mice eyes and compared it with some other contemporary AAVs. Three different AAV vectors (single strand AAV2-ssAAV2, scAAV2 and Anc80) carrying eGFP reporter gene driven by CMV promoter. The viral vectors were injected into wild type C57Bl/6 mouse eyes either via anterior chamber injection at a dose of 1×109 particles, or via intravitreal injection at a dose of 2×109 particles. At day 28 post- injection, mice eyeballs were collected and sectioned to examine GFP expression in the anterior segment by using confocal microscopy. Our data shows all three AAV vectors can transduce cornea, trabecular meshwork, and ciliary body by anterior chamber injection, as determined by GFP expression level and expression area in the cornea, Anc80 > ssAAV2 > scAAV2, and in the trabecular meshwork and ciliary body, Anc80 > scAAV2 > ssAAV2. Via intravitreal injection, GFP expression was detected in cornea and ciliary body in the Anc80 group, while no GFP expression in cornea or ciliary body was found in the ssAAV2 group. In Conclusion, anterior chamber or intravitreal injections of Anc80 efficiently transduce all components of the mouse anterior segment. These results will inform future studies that aim to deliver therapeutics in such diseases as glaucoma, which affect the cornea, iris, or ciliary body.

711. Development of a Clinically-Acceptable Lentiviral Vector for Cystic Fibrosis Airway Gene Therapy

Background: Lentiviral (LV) vectors are a promising gene delivery vehicle for cystic fibrosis airway gene therapy. However, the development of a clinically-acceptable method suited to large-scale lentivirus production remains a barrier to the translation of gene therapy to the clinic. Current virus production methods have limited-scalability and often result in contamination of the vector preparation with potentially immunogenic components such as bacterially-derived plasmid DNA, and animal sera products. Accordingly, a scalable process for the production of clinical-grade vector is required.Methods: LV vector preparations were treated with Deoxyribonuclease I (DNase I) to remove residual plasmid DNA, with the effectiveness measured by PCR using primers to detect the presence of the cytomegalovirus promoter sequence. The potential for scalable LV vector production in serum-free conditions was investigated by performing transient transfection of HEK 293FT cells adapted to grow in serum-free, suspension culture. To determine the efficiency of the virus production system RNA titering was performed using a commercial qPCR kit, and reporter gene expression of GFP and Luciferase were quantified using FACS and IVIS bioluminescence imaging.Results: Plasmid DNA was detected in the vector preparations following DNase treatment, indicating that DNase I was ineffective at eliminating the residual plasmid DNA. Lentivirus was successfully produced using transient transfection of serum-free, suspension growing HEK 293FT cells, and was scaled-up to a one-litre culture capacity in shake flasks. Titres of up to 108 TU/mL were achieved in the suspension cell culture system, which is comparable to the yields obtained in the standard adherent culture system.Conclusions: The protocol developed in this study for serum-free suspension culture virus production resulted in high vector titres that were comparable to those routinely achieved using adherent cells. Furthermore, relative to traditional virus production methods, this protocol is easily scalable, less labour intensive, and has less potential for contamination of the final vector preparation. DNase I treatment was ineffective at removing residual plasmid DNA from the vector preparation, indicating that enzymatic removal of plasmid DNA may not be a feasible approach.Funding: The University of Adelaide; Cure4CF Foundation.

756. Stable Amelioration of Hemophilia B in Dogs by Intravenous Administration of Lentiviral Vectors Expressing Hyper-Functional Factor IX

Lentiviral vectors (LVs) are attractive vehicles for liver-directed gene therapy by virtue of their ability to stably integrate in the genome of target cells and the low prevalence of pre-existing immunity against HIV in humans. Over the past years, we have developed a LV platform that can achieve stable transgene expression in the liver, induce transgene-specific immune tolerance and establish correction of hemophilia in mouse models upon systemic administration. This LV is designed to stringently target transgene expression to hepatocytes through transcriptional and microRNA-mediated regulation. We then investigated the efficacy and safety profile of portal vein administration of LVs expressing canine factor IX (FIX) in a canine model of hemophilia B. We produced large-scale batches of LVs qualified for in vivo administration and treated adult hemophilia B dog by portal vein administration. We observed long-term stable reconstitution of canine FIX activity up to 1% of normal and significant amelioration of the clinical phenotype in 3 treated dogs with 6, 3.5 and 2.5 years of follow up. LV infusion was associated with transient signs of inflammatory response and mild hepatotoxicity, which could be abrogated by pretreatment with anti-inflammatory drugs. There was no detectable long-term toxicity or development of FIX inhibitors. In the perspective of clinical translation and to increase therapeutic efficacy, we next treated two 10-kg hemophilia B dogs by peripheral vein administration of LVs expressing a codon-optimized and hyperfunctional canine FIX at a 5-fold higher dose than those previously administered. Intravenous LV administration was well tolerated with mild and self-limiting elevation of aminotransferases in one dog. In the dog that reached more than 1 year of follow up FIX activity ranged between 4-8% of normal. Treatment of two more dogs at a higher dose is underway. Overall, our studies position LV-mediated liver gene therapy for further pre-clinical development and clinical translation. LVs may thus complement other available vectors to address some of the outstanding challenges posed by liver gene therapy of hemophilia and conceivably other diseases.

7. High-Throughput Sequencing of AAV Proviral Libraries from the Human Population Reveals Novel Variants with Unprecedented Intra-and Inter-Tissue Diversity

Recombinant adeno-associated viruses (rAAVs) remain the most promising gene delivery vehicle for human gene therapy. The development of novel rAAVs with high transduction efficiency and specific tropisms primarily relies on PCR cloning of natural variants from primate tissues, or the engineering of capsids via directed evolution and rational design. Faithful clinical translation of leading therapeutic AAV serotypes derived from non-human tissues remains a hot-button issue. Here, we employed high-throughput variant profiling of natural AAV proviral libraries from the human population to identify novel AAV variants with unique tissue tropisms. We screened 844 surgical specimens, encompassing a wide range of tissue types and disease states from 455 patients by robust signature PCR. We found that >80% of human tissues are AAV positive with notable serotype frequencies (AAV2/3 chimera > AAV8 > AAV2 > AAV6) distributed among different tissues. Notably, we also used qPCR primers against the conserved AAV rep sequence to quantify the abundance of pAAV proviral genomes in human tissues. Next, to ensure the highest return on identifying novel variants, we employed single-molecule, real-time (SMRT) sequencing to characterize full-length rep and cap sequences. Our preliminary findings from a single patient tissue sample revealed more than 800 unique DNA sequence variants (originating from single nucleotide polymorphisms, genetic recombination, and de novo mutations) that account for more than 50 unique amino acid sequence variants. We also detected significant inter-patient variation, as well as high inter-tissue variability within individual patients. These findings suggest that AAV exhibits the capacity for high genomic diversity and accelerated intra-host evolution. These characteristics, which are shared among many other viruses, are presumed to promote survival against the host immune response, and fitness in tissue-specific cellular environments.We also validated a selection of AAV8 variants by assessing their capacity for vector packaging and in vivo gene transfer. We found that a subset of variants has a stronger competency for transcytosis and tropism to liver and muscle than the conventional AAV8 serotype. Remarkably, one AAV8 variant can cross the blood-brain barrier and target the CNS more efficiently than AAV9 after intravenous injection of adult mice. Another noteworthy discovery is the sequence variability found in the assembly-activating protein (AAP) gene, which is encoded by an alternative open reading frame of the cap gene. The diversity within AAP may define the efficacy of AAV packaging among variants, and implicate its role in AAV evolution and fitness.By exploiting AAV-host tissues as a natural incubator for viral evolution to potentiate new AAV capsid sequences, and employing a strategy to profile intact capsid sequences on a high-throughput scale, this study highlights the unprecedented diversity and evolutionary capacity of AAV. Importantly, these new findings reshape the versatility of rAAV as the ideal biotherapy for targeting a range of normal and/or diseased tissues.

286. Genome Editing of Inducible Cell Lines for Scalable Production of Improved Lentiviral Vectors for Human Gene Therapy

Lentiviral vectors (LVs) represent efficient and versatile vehicles for gene therapy. Current manufacturing of clinical-grade LVs mostly relies on transient transfection of plasmids expressing the multiple vector components. This method is labor and cost intensive and becomes challenging when facing the need of scale-up and standardization. The development of stable LV producer cell lines will greatly facilitate overcoming these hurdles. We have generated an inducible LV packaging cell line, carrying the genes encoding for third-generation vector components stably integrated in the genome under the control of tetracycline-regulated promoters. These LV packaging cells are stable in culture even after single-cell cloning and can be scaled up to large volumes. In order to minimize the immunogenicity of LVs for in vivo administration, we set out to remove the highly polymorphic class-I major histocompatibility complexes (MHC-I) expressed on LV packaging cells and incorporated in the LV envelope. We performed genetic disruption of the β-2 microglobulin (B2M) gene, a required component for the assembly and trafficking of all MHC-I to the plasma membrane in LV producer cells, exploiting the RNA-guided Cas9 nuclease. The resulting B2M-negative cells were devoid of surface-exposed MHC-I and produced MHC-free LVs. These LVs retain their infectivity on all tested cells in vitro and efficiently transduced the mouse liver upon intravenous administration. Strikingly, the MHC-free LVs showed significantly reduced immunogenicity in a T-cell activation assay performed on human primary T cells co-cultured with syngeneic monocytes exposed to LV, from several (n=7) healthy donors. To reproducibly generate LV-producer cell lines from these cells, we insert the LV genome of interest in the AAVS1 locus, chosen for robust expression, exploiting engineered nucleases and homology-directed repair. By this strategy, we have obtained several independent producer cell lines for LVs that express marker or therapeutic genes and are devoid of plasmid DNA contamination. LVs produced by these cells reproducibly show titer and infectivity within the lower bound range of standard optimized transient transfection, and effectively transduce relevant target cells, such as hematopoietic stem/progenitor cells and T cells ex vivo and the mouse liver in vivo. Overall, we provide evidence that rationally designed targeted genome engineering can be used to improve the yield, quality, safety and sustainability of LV production for clinical use.

672. Hybrid AAV/Phage Vector Enhances Chemotherapy Efficacy Against Cancer

Chemotherapy has been widely used for cancer treatment, both in early and late stages. However, chemotherapy does not selectively target tumor cells, as normal cells are also harmed by the drugs resulting in several side effects. Another major obstacle to the success of chemotherapy in cancer treatment is the development of tumor drug resistance by cancer cells. In order to avoid these problems, the combination of chemotherapy with other therapeutic strategies has been used in order to lower the chemotherapeutic drug dose. Gene therapy is one of the therapeutic strategies that can be combined with chemotherapy. Mammalian viruses are well recognized vehicles for gene therapy, but major drawbacks of these viral vectors are broad tissue tropism following systemic administration (low specificity for the target cells/tissues), their fragility to harsh environments and difficulty for large scale production. Therefore, our group has developed a vector from bacteriophage, a bacteria virus also named phage. This novel engineered phage, called AAV/Phage, displays the RGD4C peptide to target a specific receptor (αvβ3 integrin) on cancer cell surface, while the phage genome is merged with recombinant rAAV2 virus genome carrying the transgene to deliver. In comparison with mammalian viral vectors, the production of AAV/Phage is quicker, simpler and more economical. In addition, the vector is stable at 4oC for many years. The AAV/Phage vector efficiently targeted, delivered, and expressed transgene in cancer cells in vitro. We also proved that the vector selectively targeted gene delivery to tumors after intravenous injection in animal models of cancer. Moreover, in brain tumor models, the vector can penetrate through blood brain barrier and selectively delivers transgene expression to brain tumors. We used our vector as combination therapies with some well-known cancer drugs, such as doxorubicin, and temozolomide. Combination treatment of AAV/Phage vector carrying the Herpes Simplex virus thymidine kinase gene (AAV/Phage-HSVtk.) with doxorubicin increased the targeted cancer cell killing in 2D tissue cultures and 3D tumor spheroids of rat gliosarcoma (9L) and human melanoma (M21) cells. We found that this increase in tumor cell killing was associated with a synergistic effect of doxorubicin on enhancing gene expression by AAV/Phage. We then combined AAV/Phage carrying short hairpin RNA to suppress mTOR gene expression (AAV/Phage-shmTOR) with temozolomide to treat medulloblastoma cells (DAOY). The results exhibited that treatment of medulloblastma with the vector alone efficiently suppresses the expression of mTOR gene, but has no effect on cell killing. Treatment of temozolomide at low dose (500 uM) did not have effect on cell killing, but combination therapies of temozolomide with AAV/Phage-shmTOR significantly increased cell death. Altogether, our results demonstrate that combination of AAV/Phage carrying therapeutic genes with cancer chemotherapeutic drugs is an effective strategy for cancer treatment. In future work, we plan to investigate the efficacy of AAV/Phage and cancer drug combination treatment in pre-clinical models of cancer.

Recent Patents in Oncolytic Virotherapy.

Recent innovative and advanced developments in the diagnosis and treatment of human diseases as well as enhanced in-depth understanding of virus molecular biology have opened novel avenues with respect to the patent landscape. Included are viruses utilized in the development of anticancer agents, agents that are employed against the spread of infectious viral diseases, RNA silencing agents and virus-derived expression vectors that can be used for over-expression of therapeutic proteins or as gene therapy vehicles. The current review describes several recent patents pertaining to virus sequences and their medical and biotechnological applications.

Genetic Manipulation of Brown Fat Via Oral Administration of an Engineered Recombinant Adeno-associated Viral Serotype Vector.

Recombinant adeno-associated virus (rAAV) vectors are attractive vehicles for gene therapy. Gene delivery to the adipose tissue using naturally occurring AAV serotypes is less successful compared to liver and muscle. Here, we demonstrate that oral administration of an engineered serotype Rec2 led to preferential transduction of brown fat with absence of transduction in the gastrointestinal track. Among the six natural and engineered serotypes being compared, Rec2 was the most efficient serotype achieving high level transduction at a dose 1~2 orders lower than reported doses for systemic administration. Overexpressing vascular endothelial growth factor (VEGF) in brown fat via oral administration of Rec2-VEGF vector increased the brown fat mass and enhanced thermogenesis. In contrast, knockdown VEGF in brown fat of VEGF (loxP) mice via Rec2-Cre vector hampered cold response and decreased brown fat mass. Oral administration of Rec2 vector provides a novel tool to genetically manipulate brown fat for research and therapeutic applications.

Viral Vectors for Gene Therapy: Translational and Clinical Outlook.

In a range of human trials, viral vectors have emerged as safe and effective delivery vehicles for clinical gene therapy, particularly for monogenic recessive disorders, but there has also been early work on some idiopathic diseases. These successes have been enabled by research and development efforts focusing on vectors that combine low genotoxicity and immunogenicity with highly efficient delivery, including vehicles based on adeno-associated virus and lentivirus, which are increasingly enabling clinical success. However, numerous delivery challenges must be overcome to extend this success to many diseases; these challenges include developing techniques to evade preexisting immunity, to ensure more efficient transduction of therapeutically relevant cell types, to target delivery, and to ensure genomic maintenance. Fortunately, vector-engineering efforts are demonstrating promise in the development of next-generation gene therapy vectors that can overcome these barriers. This review highlights key historical trends in clinical gene therapy, the recent clinical successes of viral-based gene therapy, and current research that may enable future clinical application.

OP043D CULTURE OF MURINE NEURAL STEM CELLS ON DECELLULARIZED MOUSE BRAIN SECTIONS

INTRODUCTION: Their inherent tumour tropism renders neural stem cells (NSC) intriguing as tumour-targeting gene therapy vehicles for brain cancers. However, effective cell therapy relies on the intrinsic capacity of cellular grafts to overcome hypoxic and/or immunological barriers during transplantation. In this context, it is hypothesized that structural support for grafted NSC - in the form of a 3D biocompatible matrix - will be of utmost importance. METHOD: A protocol to decellularize 1.5 mm thick mouse brain sections was designed and validated using molecular analysis and microscopy. Next, the resulting extracellular matrix (ECM) scaffolds were seeded with murine NSC expressing both eGFP and luciferase. Bioluminescence imaging was used to monitor survival and growth. Phenotype was evaluated using confocal microscopy and compared to standard 2D cultured NSC. RESULTS: We optimised and validated a novel decellularization protocol for 1.5 mm thick mouse brain sections, resulting in the generation of acellular 3D brain tissue-derived ECM scaffolds. These 3D ECM scaffolds supported long-term ex vivo survival and growth of NSC during a 2-13 week monitoring period. Confocal microscopy revealed the 3D organisation of scaffold-supported NSC cultures. Under mitogenic stimuli, most cells in this 3D culture retained the NSC phenotype, with only a small minority undergoing spontaneous differentiation. CONCLUSION: The development of this new 3D NSC culture system will become useful for new research on the development of neural networks, brain tumour remodelling, drug testing and brain cell transplantation research. Moreover, it may offer a new modality for 3D in vitro brain research when using brain cancer stem cells.

IL-21-Expressing Mesenchymal Stem Cells Prevent Lethal B-Cell Lymphoma Through Efficient Delivery of IL-21, Which Redirects the Immune System to Target the Tumor

Interleukin (IL)-21, a proinflammatory cytokine, has been developed as an immunotherapeutic approach due to its effects on various lymphocytes, including natural killer (NK) cells and T cells; however, the clinical success in cancer patients has been limited. Recently, mesenchymal stem cells (MSCs) have emerged as vehicles for cancer gene therapy due to their inherent migratory abilities toward tumors. In the present study, we hypothesized that MSCs, genetically modified to express high levels of IL-21 (IL-21/MSCs), can enhance antitumor responses through localized delivery of IL-21. For tumor induction, BALB/c mice were injected intravenously with syngeneic A20 B-cell lymphoma cells to develop a disseminated B-cell lymphoma model. Then, 6 days following tumor induction, the tumor-bearing mice were treated with IL-21/MSCs weekly, four times. Systemic infusion of A20 cells led to hind-leg paralysis as well as severe liver metastasis in the control group. The IL-21/MSC-treated group showed delayed tumor incidence as well as improved survival, whereas the MSC- and recombinant adenovirus-expressing IL-21 (rAD/IL-21)-treated groups did not show significant differences from the untreated mice. These therapeutic effects were associated with high levels of IL-21 delivered to the liver, which prevented the formation of tumor nodules. Furthermore, the infusion of IL-21/MSCs led to induction of effector T and NK cells, while potently inhibiting immune suppressor cells. Our findings demonstrate that IL-21-expressing MSCs have the therapeutic potential to induce potent antitumor effects against disseminated B-cell lymphoma through localized IL-21 delivery and induction of systemic antitumor immunity.

Adenoviral Vectors in Gene Therapy

This review is focused on gene therapy, especially adenovirus vectors and their relationship with the immune system response. Adenovirus vectors belong to the most used gene delivery vehicles in gene therapy, study of gene expression or immunotherapy. One of the most important questions concerning their use is their influence on organism in vivo. Study of immunomodulating properties of the adenovirus vectors opens a way for further manipulation and their more effective practical use.

PAMAM Dendrimer Conjugated with Cell‐penetrating Peptide‐derived Oligopeptides for Enhanced Cell Uptake and Gene Delivery

Cell‐penetrating peptides (CPPs), originated from membrane translocating sequences of viruses, can deliver biomolecules effectively into cells. Research on natural and synthetic CPPs, for delivering drugs and genes reveals that this process requires cellular uptake independent of receptors and endosomes to minimize damage to the cell membrane. To this end, we conjugated CPP‐derived oligopeptides to the surface of polyamidoamine (PAMAM) dendrimers to develop novel nanocarriers that improve cellular uptake transfection efficiency. PAMAM generation 4 (G4) was conjugated with oligopeptides composed of lysine and phenylalanine (KFF, FFK). The DNA condensation efficiency of PAMAM G4‐KFF and PAMAM G4‐FFK was determined using an agarose gel retardation assay. The sizes of the polyplexes were determined using dynamic light scattering as well as scanning and transmission electron microscopy. Cytotoxicity and transfection efficiency were evaluated using the HeLa and HepG2 cells. The transfection efficiencies of PAMAM G4‐KFF and PAMAM G4‐FFK were comparable with that of the gold‐standard transfection reagent polyethylenimine (PEI25 kDa). These results suggest that these nanocarriers will serve as efficient non‐viral vehicles for gene therapy.

Mesenchymal Stem Cells Overexpressing Angiotensin-Converting Enzyme 2 Rescue Lipopolysaccharide-Induced Lung Injury

Bone marrow-derived mesenchymal stem cells (MSCs), which have beneficial effects in acute lung injury (ALI), can serve as a vehicle for gene therapy. Angiotensin-converting enzyme 2 (ACE2), a counterregulatory enzyme of ACE that degrades angiotensin (Ang) II into Ang 1-7, has a protective role against ALI. Because ACE2 expression is severely reduced in the injured lung, a therapy targeted to improve ACE2 expression in lung might attenuate ALI. We hypothesized that MSCs overexpressing ACE2 would have further benefits in lipopolysaccharide (LPS)-induced ALI mice, when compared with MSCs alone. MSCs were transduced with ACE2 gene (MSC-ACE2) by a lentiviral vector and then infused into wild-type (WT) and ACE2 knockout (ACE2(-/y)) mice following an LPS-induced intratracheal lung injury. The results demonstrated that the lung injury of ALI mice was alleviated at 24 and 72 h after MSC-ACE2 transplantation. MSC-ACE2 improved the lung histopathology and had additional anti-inflammatory effects when compared with MSCs alone in both WT and ACE2(-/y) ALI mice. MSC-ACE2 administration also reduced pulmonary vascular permeability, improved endothelial barrier integrity, and normalized lung eNOS expression relative to the MSC group. The beneficial effects of MSC-ACE2 could be attributed to its recruitment into the injured lung and enhanced local expression of ACE2 protein without changing the serum ACE2 levels after MSC-ACE2 transplantation. The biological activity of the increased ACE2 protein decreased the Ang II amount and increased the Ang 1-7 level in the lung when compared with the ALI and MSC-only groups, thereby inhibiting the detrimental effects of accumulating Ang II. Therefore, compared to MSCs alone, the administration of MSCs overexpressing ACE2 resulted in a further improvement in the inflammatory response and pulmonary endothelial function of LPS-induced ALI mice. These additional benefits could be due to the degradation of Ang II that accompanies the targeted overexpression of ACE2 in the lung.

Bone marrow-derived mesenchymal stem cells co-expressing interleukin-18 and interferon-β exhibit potent antitumor effect against intracranial glioma in rats.

Bone marrow-derived mesenchymal stem cells (BMSCs) are promising gene vehicles for cancer gene therapy. In our previous study, we reported that BMSCs expressing interleukin (IL)-18 effectively inhibit the growth of glioma in rats. In the present study, we further detected the effect of BMSCs co-expressing IL-18 and interferon (IFN)-β, both of which are immunostimulatory cytokines. BMSCs were genetically engineered to express IL-18 and IFN-β by transfection of recombinant lentivirus-mediated gene transfer. Results showed that BMSCs co-expressing the two cytokines displayed more significant inhibition effect on glioma cell growth invitro when compared with BMSCs solely expressing IL-18 or IFN-β. Treatment of BMSCs co-expressing IL-18 and IFN-β significantly prolonged the survival and inhibited tumor growth in a rat intracranial glioma model. Furthermore, these genetically engineered BMSCs remarkably promoted cell apoptosis, antitumor cytokine production and CD4+ and CD8+ T-cell infiltration in intracranial glioma tissues than BMSCs solely expressing IL-18 or IFN-β. Results of the present study suggested that IL-18 and IFN-β had a synergistic effect on glioma inhibition. Moreover, results provided evidence that delivery of IL-18 and IFN-β by BMSCs may be an excellent and promising approach to develop an effective treatment protocol for glioma therapy.

Human Adipose Tissue-Derived Mesenchymal Stem Cells Target Brain Tumor-Initiating Cells.

In neuro-oncology, the biology of neural stem cells (NSCs) has been pursued in two ways: as tumor-initiating cells (TICs) and as a potential cell-based vehicle for gene therapy. NSCs as well as mesenchymal stem cells (MSCs) have been reported to possess tumor tropism capacities. However, there is little data on the migratory capacity of MSCs toward brain tumor-initiating cells (BTICs). This study focuses on the ability of human adipose tissue derived MSCs (hAT-MSCs) to target BTICs and their crosstalk in the microenvironment. BTICs were isolated from three different types of brain tumors. The migration capacities of hAT-MSCs toward BTICs were examined using an in vitro migration assay and in vivo bioluminescence imaging analysis. To investigate the crosstalk between hAT-MSCs and BTICs, we analyzed the mRNA expression patterns of cyto-chemokine receptors by RT-qPCR and the protein level of their ligands in co-cultured medium. The candidate cyto-chemokine receptors were selectively inhibited using siRNAs. Both in vitro and in vivo experiments showed that hAT-MSCs possess migratory abilities to target BTICs isolated from medulloblastoma, atypical teratoid/rhabdoid tumors (AT/RT) and glioblastoma. Different types of cyto-chemokines are involved in the crosstalk between hAT-MSCs and BTICs (medulloblastoma and AT/RT: CXCR4/SDF-1, CCR5/RANTES, IL6R/IL-6 and IL8R/IL8; glioblastoma: CXCR4/SDF-1, IL6R/IL-6, IL8R/IL-8 and IGF1R/IGF-1). Our findings demonstrated the migratory ability of hAT-MSCs for BTICs, implying the potential use of MSCs as a delivery vehicle for gene therapy. This study also confirmed the expression of hAT-MSCs cytokine receptors and the BTIC ligands that play roles in their crosstalk.

422. In a Suicide Gene Therapy Approach to Cancer, γ-Irradiation Can Completely Abolish the Bystander Effect of the PA-STK Ovarian Tumour Cell Line Due to Its Radiation Sensitivity, Resulting in Necrotic Cell Death

The effect of irradiation on HSV-TK-expressing tumour cells and their bystander killing has been examined. The data presented shows that the irradiation substantially reduced the ability of PA-STK ovarian cells to induce the bystander killing of unmodified tumour cells. PA-STK cells were found to be very sensitive to irradiation and appeared to die too rapidly (even after a low dose of 500-rad) to enable metabolic conversion of GCV to its phosphorylated products, and/or fail to form gap-junctions with their neighbouring cells. Characterisation of tumour cell death showed that PA-STK cells undergo picnosis (a form of necrosis) after irradiation. In contrast, the TK-modified human and mouse mesothelioma cells were found to retain their in vitro bystander killing effect after irradiation. These results suggest that PA-STK cells are not suitable for clinical trials if the cells need to be lethally irradiated before in vivo use. However, the human mesothelioma cell line CRL-5830-TK appears to retain its bystander killing potential after lethal irradiation. In clinical trials, it may therefore be a suitable vehicle for HSV-TK suicide gene therapy for mesothelioma. The low IC50 of GCV in these cells (about 1μμ) makes them particularly attractive.

28. Intravenous Administration of Lentiviral Vectors Expressing Hyperactive Factor IX Converts Severe Into Mild Hemophilia B in a Canine Model

Lentiviral vectors (LVs) are attractive vehicles for liver-directed gene therapy by virtue of their ability to stably integrate in the genome of target cells and the lack of pre-existing immunity against vector components in most humans. Over the past years, we have developed a LV platform that can achieve stable transgene expression in the liver, induce transgene-specific immune tolerance and establish correction of hemophilia in mouse models upon systemic administration. This LV is designed to stringently target transgene expression to hepatocytes through transcriptional and microRNA-mediated regulation. We then investigated the efficacy and safety profile of portal vein administration of LVs expressing wild-type, codon-optimized (c.o.) or c.o. and hyperactive factor IX (FIX) in a canine model of hemophilia B. We produced large-scale batches of LVs qualified for in vivo administration and treated adult hemophilia B dog by portal vein administration. We observed long-term stable reconstitution of canine FIX activity up to 1% of normal and significant amelioration of the clinical phenotype in 3 treated dogs (>9 years cumulative follow up). LV infusion was associated with transient signs of inflammation and mild hepatotoxicity, which could be abrogated by pretreatment with anti-inflammatory drugs. There was no detectable long-term toxicity or development of FIX inhibitors. In the perspective of clinical translation and to increase therapeutic efficacy, we next treated an 11-kg, hemophilia B dog by peripheral vein administration of LVs expressing the c.o. and hyperactive canine FIX at a 5-fold higher dose than those previously administered. At the current follow-up (3 months after gene therapy) FIX activity is 6-9% of normal. Intravenous LV administration, coupled with a 1-day anti-inflammatory and anti-histamine pre-treatment, induced mild and selflimiting leukopenia and elevation of aminotransferases. Treatment of more hemophilia B dogs is underway to confirm and extend these results. Overall, our studies, which suggest comparable efficacy of LV by both portal and peripheral vein administration, position LV-mediated liver gene therapy for further pre-clinical development and clinical translation. LVs may thus complement other available vectors to address some of the outstanding challenges posed by liver gene therapy of hemophilia and conceivably other diseases.

6. Targeted Genome Editing of Cell Lines for Improved and Scalable Production of Lentiviral Vectors for Human Gene Therapy

Lentiviral vectors (LVs) represent efficient and versatile vehicles for gene therapy. The manufacturing of clinical-grade LVs relies on transient transfection of vector components. This method is labor and cost intensive and becomes challenging when facing the need of scale-up and standardization. The development of stable LV producer cell lines will greatly facilitate overcoming these hurdles. We have generated an inducible LV packaging cell line, carrying the genes encoding for third-generation vector components stably integrated in the genome under the control of tetracycline-regulated promoters. In order to minimize the immunogenicity of LVs for in vivo administration, we set out to remove the highly polymorphic and antigenic class-I major histocompatibility complex (MHC-I) expressed on LV packaging cells and subsequently incorporated on the LV envelope. We performed genetic disruption of the β-2 microglobulin (B2M) gene, a required component for the assembly and trafficking of the MHC-I to the plasma membrane in LV producer cells, exploiting the RNA-guided Cas9 nuclease. We generated B2M-negative cells devoid of surface-exposed MHC-I, which retain the ability to produce LVs. In order to insert the LV genome of interest in the packaging cell line, we performed site-specific integration in predetermined loci of the genome of these cells, chosen for robust expression, exploiting artificial nucleases and homology-directed repair. In several independent iterations of this process, we generated producer cell lines both for LV expressing marker genes and a therapeutic gene, i.e. coagulation factor IX (FIX), the gene mutated in hemophilia B. We show that these LV producer cells are stable in culture and can produce several liters of LV-containing conditioned medium. These LVs have comparable or only slightly lower infectious titer and specific infectivity than LVs produced by state-of-the-art transient transfection process and can transduce therapeutically relevant target cells, such as hematopoietic stem/progenitor cells and T lymphocytes to high efficiency. Moreover, we intravenously administered FIX-expressing LVs produced by the cell line to hemophilia B mice and established therapeutic levels of circulating FIX. These data indicate that site-specific integration is an efficient, rapid and reproducible method to generate LV producer cells, starting from a universal stable inducible LV packaging cell line. Overall, we provide evidence that rationally designed targeted genome engineering can be used to improve the quality, safety and sustainability of LV production for clinical use.

Irradiation-induced localization of IL-12-expressing mesenchymal stem cells to enhance the curative effect in murine metastatic hepatoma.

Irradiation in conjunction with gene therapy is considered for efficient cancer treatment. Mesenchymal stem cells (MSCs), due to their irradiation-promotable tumor tropism, are ideal delivery vehicles for gene therapy. In this study, we investigated whether treatment with radiation and interleukin (IL)-12-expressing MSCs (MSCs/IL-12) exerts improved antitumor effects on murine metastatic hepatoma. HCa-I and Hepa 1-6 cells were utilized to generate heterotopic murine hepatoma models. Tumor-bearing mice were treated with irradiation or MSCs/IL-12 alone, or a combination. Monocyte chemoattractant protein-1 (MCP-1/CCL2) expression was assessed in irradiated hepatoma tissues to confirm a chemotactic effect. Combination treatment strategies were established and their therapeutic efficacies were evaluated by monitoring tumor growth, metastasis and survival rate. IL-12 expression was assessed and the apoptotic activity and immunological alterations in the tumor microenvironment were examined. MCP-1/CCL2 expression and localization of MSCs/IL-12 increased in the irradiated murine hepatoma cells. The antitumor effects, including suppression of pulmonary metastasis and survival rate improvements, were increased by the combination treatment with irradiation and MSCs/IL-12. IL-12 expression was increased in tumor cells, causing proliferation of cluster of differentiation 8(+) T-lymphocytes and natural killer cells. The apoptotic activity increased, indicating that the cytotoxicity of immune cells was involved in the antitumor effect of the combined treatment. Treatment with irradiation and MSCs/IL-12 showed effectiveness in treating murine metastatic hepatoma. IL-12-induced proliferation of immune cells played an important role in apoptosis of tumor cells. Our results suggest that treatment with irradiation and MSCs/IL-12 may be a useful strategy for enhancing antitumor activity in metastatic hepatoma.