Therapeutic Transgene Research Articles

Ex vivo Generation of Genetically Modified Macrophages from Human Induced Pluripotent Stem Cells

Background: Pluripotent stem cells, including induced pluripotent stem cells (iPSCs), have the capacity to differentiate towards all three germ layers and have been highlighted as an attractive cell source for the field of regenerative medicine. Thus, stable expression of therapeutic transgenes in iPSCs, as well as thereof derived progeny of hematopoietic lineage, may lay the foundation for innovative cell replacement therapies. Methods: We have utilized human iPSC lines genetically modified by lentiviral vector technology or targeted integration of reporter genes to evaluate transgene expression during hematopoietic specification and differentiation towards macrophages. Results: Use of lentiviral vectors equipped with an ubiquitous chromatin opening element (CBX3-UCOE) as well as zinc finger nuclease-mediated targeting of an expression cassette into the human adeno-associated virus integration site 1 (AAVS1) safe harbor resulted in stable transgene expression in iPSCs. When iPSCs were differentiated along the myeloid pathway into macrophages, both strategies yielded sustained transgene expression during the hematopoietic specification process including mature CD14+ and CD11b+ macrophages. Conclusion: Combination of human iPSC technology with either lentiviral vector technology or designer nuclease-based genome editing allows for the generation of transgenic iPSC-derived macrophages with stable transgene expression which may be useful for novel cell and gene replacement therapies.

Comparison of Serum rAAV Serotype-Specific Antibodies in Patients with Duchenne Muscular Dystrophy, Becker Muscular Dystrophy, Inclusion Body Myositis, or GNE Myopathy

Recombinant adeno-associated virus (rAAV) is a commonly used gene therapy vector for the delivery of therapeutic transgenes in a variety of human diseases, but pre-existing serum antibodies to viral capsid proteins can greatly inhibit rAAV transduction of tissues. Serum was assayed from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), inclusion body myositis (IBM), and GNE myopathy (GNE). These were compared to serum from otherwise normal human subjects to determine the extent of pre-existing serum antibodies to rAAVrh74, rAAV1, rAAV2, rAAV6, rAAV8, and rAAV9. In almost all cases, patients with measurable titers to one rAAV serotype showed titers to all other serotypes tested, with average titers to rAAV2 being highest in all instances. Twenty-six percent of all young normal subjects (<18 years old) had measurable rAAV titers to all serotypes tested, and this percentage increased to almost 50% in adult normal subjects (>18 years old). Fifty percent of all IBM and GNE patients also had antibody titers to all rAAV serotypes, while only 18% of DMD and 0% of BMD patients did. In addition, serum-naïve macaques treated systemically with rAAVrh74 could develop cross-reactive antibodies to all other serotypes tested at 24 weeks post treatment. These data demonstrate that most DMD and BMD patients should be amenable to vascular rAAV-mediated treatment without the concern of treatment blockage by pre-existing serum rAAV antibodies, and that serum antibodies to rAAVrh74 are no more common than those for rAAV6, rAAV8, or rAAV9.

Lentiviral Hematopoietic Stem Cell Gene Therapy in Inherited Immune and Lysoso- mal Enzyme Deficiencies

Rare diseases affect millions of people worldwide. Many of those are inherited disorders resulting in chronic disability and requiring cost-intensive care. Hematopoietic stem cell gene therapy has been developed over more than 20 years. At the state of the art, gene therapy is within reach for diseases in which (i) the genetic defect is identified, (ii) the diagnosis is made sufficiently early for a meaningful therapeutic intervention, (iii) a specific animal model is available for efficacy and safety evaluation. Appropriate therapeutic transgenes should also comply with certain biological criteria. Third-generation lentiviral vectors have been made self-inactivating (SIN) by deletion of enhancer regions from the LTR sequences thus reducing the risk of influencing nearby genes, resulting in favorable safety profiles. At the present time, lentiviral hematopoietic stem cell gene therapy has entered the stage of initial clinical implementation for immune deficiencies and lysosomal storage disorders. We discuss initial clinical trials using these vectors for selected metabolic storage disorders, which include adrenoleukodystrophy, metachromatic leukodystrophy, Hurler (MPS I), Pompe (GSD II), and Fabry diseases. This brief review summarizes the development and current clinical implementation of these approaches.

Self-adjusting synthetic gene circuit for correcting insulin resistance.

By using tools from synthetic biology, sophisticated genetic devices can be assembled to reprogram mammalian cell activities. Here, we demonstrate that a self-adjusting synthetic gene circuit can be designed to sense and reverse the insulin-resistance syndrome in different mouse models. By functionally rewiring the mitogen-activated protein kinase (MAPK) signalling pathway to produce MAPK-mediated activation of the hybrid transcription factor TetR-ELK1, we assembled a synthetic insulin-sensitive transcription-control device that self-sufficiently distinguished between physiological and increased blood insulin levels and correspondingly fine-tuned the reversible expression of therapeutic transgenes from synthetic TetR-ELK1-specific promoters. In acute experimental hyperinsulinemia, the synthetic insulin-sensing designer circuit reversed the insulin-resistance syndrome by coordinating expression of the insulin-sensitizing compound adiponectin. Engineering synthetic gene circuits to sense pathologic markers and coordinate the expression of therapeutic transgenes may provide opportunities for future gene- and cell-based treatments of multifactorial metabolic disorders.

β-cell-mimetic designer cells provide closed-loop glycemic control.

Chronically deregulated blood-glucose concentrations in diabetes mellitus result from a loss of pancreatic insulin-producing β cells (type 1 diabetes, T1D) or from impaired insulin sensitivity of body cells and glucose-stimulated insulin release (type 2 diabetes, T2D). Here, we show that therapeutically applicable β-cell-mimetic designer cells can be established by minimal engineering of human cells. We achieved glucose responsiveness by a synthetic circuit that couples glycolysis-mediated calcium entry to an excitation-transcription system controlling therapeutic transgene expression. Implanted circuit-carrying cells corrected insulin deficiency and self-sufficiently abolished persistent hyperglycemia in T1D mice. Similarly, glucose-inducible glucagon-like peptide 1 transcription improved endogenous glucose-stimulated insulin release and glucose tolerance in T2D mice. These systems may enable a combination of diagnosis and treatment for diabetes mellitus therapy.

Long-Term Expression of Von Willebrand Factor Via Piggybac-Mediated Gene Transfer

von Willebrand disease (vWD), caused by a deficiency in von Willebrand factor (vWF), is the most common inherited bleeding disorder, affecting up to 1% of the population; those affected have a varied bleeding phenotype. von Willebrand factor plays two important roles in hemostasis including platelet adhesion and stability of factor VIII (FVIII). As such, patients with von Willebrand disease type 3 have no measurable vWF protein and demonstrate low levels of FVIII. To date, persistent long-term expression of full-length vWF using gene transfer strategies has not been demonstrated, largely due to the immense size of vWF cDNA (8.4 kb). Non-viral vector systems, such as piggyBac, are used increasingly in gene targeting technologies and as tools for gene transfer applications. Nonviral DNA transposons are genetic elements consisting of inverted terminal DNA repeats which in their naturally occurring configuration flank a transposase coding sequence. The transposase follows a "cut and paste" mechanism to excise the transposon from its original genomic location and insert it into a new locus. The insect derived piggyBac (PB) can be engineered to carry a therapeutic transgene between the inverted terminal repeats. Advantages of this nonviral vector system include a large transgene cassette capacity, ease of production and purification, and potential for site-specific integration. We hypothesize PB-mediated vWF gene transfer will confer long-term expression and improve the bleeding phenotype in an animal model of vWD. We engineered PB transposon to carry a human von Willebrand factor cDNA (PB vWF). When transfected with PB-vWF and a hyperactive transposase, iPB7, the hepatocarcinoma cell line, HepG2, demonstrated secretion of vWF into supernatants compared to mock transfected controls (p < 0.01). Next, we evaluated the in vivo gene transfer efficiency in von Willebrand deficient mice by hydrodynamic tail-vein injection using PB-vWF driven by the murine albumin enhancer/human alpha anti-trypsin promoter. vWF null mice received 25 or 50 micrograms each of the PB-vWF transposon and iPB7 to determine long-term expression and phenotypic correction. vWF levels were measured prior to injection and then every 4 weeks for up to 24 weeks. Results revealed therapeutic levels (on average 50% normal mice) of vWF post gene transfer with stable expression for 24 weeks in most mice. These data indicate the PB transposon vector system may provide a long-term gene transfer strategy for vWD. To evaluate phenotypic correction, a tail clip assay was performed at the end of the study. PB-vWF gene transfer resulted in at least partial bleeding phenotype correction via tail clip. Additionally, treated mice demonstrated a rescue of the FVIII activity. These data show that the PB vector can be used to deliver this large transgene expression cassette to the liver and achieve long-term expression and phenotypic correction in vivo. DisclosuresStaber:Emergent BioSolutions: Honoraria; Baxalta: Honoraria.

The Role of Conditioning in Hematopoietic Stem-Cell Gene Therapy

Gene therapy (GT) approaches based on autologous hematopoietic stem cells (HSC) corrected ex vivo have shown therapeutic benefit in a number of inherited disorders. GT bares the advantage of allowing each patient to be her/his own donor while reducing the risks of immune-mediated complications as compared with allogeneic hematopoietic stem-cell transplantation (HSCT). In order to achieve stable engraftment of HSC, patients undergoing transplantation of allogeneic or autologous HSC receive a chemotherapy- and/or radiotherapy-based preparation. With regard to HSC-GT for inherited genetic disorders, the ideal conditioning regimen should aim to contain toxicity by reducing the dosage and/or the number of chemotherapeutic agents administered, in comparison to fully myeloablative preparations employed in conventional allogeneic HSCT. To meet this aim, a profound knowledge of the disease-specific biological background and of the therapeutic transgene levels, as well as of the key principles of transplantation, are required. While low-dose conditioning is sufficient to create a mixed chimerism when gene-corrected cells are endowed with a natural selective advantage, such as in the case of immune deficiencies, myeloablative doses are necessary when high levels of engraftment are required in disease such as lysosomal storage disorders and beta thalassemia. Therefore, the intensity and type of conditioning regimen administered to patients undergoing HSC-GT should be tailored to reach a minimal efficacious therapeutic target level while sparing toxicity. Novel strategies based on monoclonal antibodies selectively depleting blood cells and associated with limited extramedullary toxicity might be successfully employed in the context of HSC-GT in the near future. This review focuses on the role of the conditioning regimen in HSC-GT, and in particular, it highlights the importance of modulating the preparative chemotherapy based on disease biology and transgene expression in order to optimize outcome.

Myeloid-derived suppressor cells expressing a self-antigen ameliorate experimental autoimmune encephalomyelitis

Previous work by our group showed that transferring bone marrow cells transduced with a self-antigen induced immune tolerance and ameliorated experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). We also found that following retroviral transduction of murine bone marrow (BM) cells, the majority of cells generated and transduced were myeloid-derived suppressor cells (MDSCs). Here, we aimed to determine whether purified antigen-expressing MDSCs have similar therapeutic effects than those of unfractionated BM, and to investigate their potential mechanisms. We performed phenotypic and functional analyses in these cells using the same animal model, and we used purified antigen-expressing MDSCs in preventive and therapeutic approaches. These cells exerted therapeutic effects similar to those of BM cells, which depended upon self-antigen expression. The majority of monocytic (M)-MDSCs expressed the immunosuppressive molecule programmed death ligand-1 (PD-L1), CD80, CD86 and MHC class II molecules. Additionally, the animals infused with antigen-expressing cells exhibited lower percentages of activated T cells and higher percentages of B cells with a regulatory phenotype (B220+CD1dhigh CD5+) in the spleen than their respective controls. MDSCs expressing self-antigens, alloantigens or therapeutic transgenes are tolerogenic and can be exploited therapeutically in autoimmune diseases, transplantation and in gene therapy, respectively.

Oncolytic vaccinia virus inhibits human hepatocellular carcinoma MHCC97-H cell proliferation via endoplasmic reticulum stress, autophagy and Wnt pathways.

Hepatocellular carcinoma (HCC) is a highly lethal malignancy. Vaccinia virus (VV) possessed many inherent advantages with respect to being engineered as a vector for cancer gene therapy, although the mechanism of action remains to be explored further. We constructed a thymidine kinase gene insertional inactivated VV, named VV-Onco, and then tested its effects on cell viability, apoptosis and colony formation ability in a highly metastatic human hepatocellular carcinoma cell line MHCC97-H, and also investigated the potential cell signal pathways involved in this action. VV-Onco induced strong cytotoxicity and apoptosis and also inhibited the colony formation of MHCC97-H cells. The tumor cell apoptosis induced by VV-Onco is likely mediated via endoplasmic reticulum stress, autophagy and Wnt signaling pathways. The downregulation of survivin and c-Myc may also play a role in VV-Onco induced cell death. The results of the present study provide new insights into the mechanisms of VV-induced tumor cell death. The engineered recombinant VV containing optimized therapeutic transgenes may represent a new avenue for cancer gene therapy. Copyright © 2016 John Wiley & Sons, Ltd.

Phase 1 Study of Intravenous Oncolytic Poxvirus (vvDD) in Patients With Advanced Solid Cancers

We have conducted a phase 1 study of intravenous vvDD, a Western Reserve strain oncolytic vaccinia virus, on 11 patients with standard treatment-refractory advanced colorectal or other solid cancers. The primary endpoints were maximum tolerated dose and associated toxicity while secondary endpoints were pharmacokinetics, pharmacodynamics, immune responses, and antitumor activity. No dose-limiting toxicities and treatment related severe adverse events were observed. The most common adverse events were grades 1/2 flu-like symptoms. Virus genomes were detectable in the blood 15–30 minutes after virus administration in a dose-dependent manner. There was evidence of a prolonged virus replication in tumor tissues in two patients, but no evidence of virus replication in non-tumor tissues, except a healed injury site and an oral thrush. Over 100-fold of anti-viral antibodies were induced in patients' sera. A strong induction of inflammatory and Th1, but not Th2 cytokines, suggested a potent Th1-mediated immunity against the virus and possibly the cancer. One patient showed a mixed response on PET-CT with resolution of some liver metastases, and another patient with cutaneous melanoma demonstrated clinical regression of some lesions. Given the confirmed safety, further trials evaluating intravenous vvDD in combination with therapeutic transgenes, immune checkpoint blockade or complement inhibitors, are warranted.

An edible switch for gene therapy.

The expression of therapeutic transgenes in mice is made responsive to the amino acid content of the diet.

Abstract 2234: Exploring the oncolytic potential of Newcastle Disease Virus

Abstract Oncolytic viruses are live, replication-competent viruses that infect and/or replicate selectively in tumour cells leading to the destruction of the infected cell. Cell death occurs as a natural consequence of the viral life cycle and newly produced virions can infect and kill neighbouring tumour cells leading to an amplified therapeutic effect. Oncolysis has the added benefit of potentially producing novel tumour antigens that can induce an immune-mediated therapeutic response. Newcastle Disease Virus (NDV) is an avian paramyxovirus, which has proven safety and demonstrated efficacy against a variety of mammalian cancers in PhI clinical studies. Using reverse genetics, we generated a recombinant strain of NDV that overcomes environmental and regulatory concerns uncoupling oncolytic potency and avian pathogenicity. We have additionally inserted the GM-CSF gene in order to maximize anti-tumour immune response and provide an in situ, patient/tumour- specific vaccine, combined with potent oncolysis. We have evaluated the biological characteristics of recNDVGM-CSF in vivo and in vitro. In vitro recNDVGM-CSF selectively replicates in and kills a wide variety of tumour cell types. In vivo, using a fibrosarcoma model that supports viral replication, a single administration (intra-tumoural or systemic) was able to cure 80% of tumour bearing mice. Additionally, following IV administration recNDVGM-CSF localises to and replicates within the tumour leading to a localised GM-CSF production within the tumour. The inherent properties of NDV (self-propagation, tumour-selective replication, immune activation) coupled with the ability to genetically engineer NDV to express therapeutic transgenes may provide a multi-modal attack on the tumour, delivering greater benefit to patients. Citation Format: James A. Harper, Jon Travers, Ruth Franks, Shannon Burke, Christel Navarro, Cheng Xing, Weijia Wang, Qi Xu, Robert W. Wilkinson, Hong Jin, Danielle Carroll. Exploring the oncolytic potential of Newcastle Disease Virus. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2234.

Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis

The Sleeping Beauty transposon (SB-Tn) system is being used widely as a DNA vector for the delivery of therapeutic transgenes, as well as a tool for the insertional mutagenesis in animal models. In order to accurately assess the insertional potential and properties related to the integration of SB it is essential to determine the copy number of SB-Tn in the host genome. Recently developed SB100X transposase has demonstrated an integration rate that was much higher than the original SB10 and that of other versions of hyperactive SB transposases, such as HSB3 or HSB17. In this study, we have constructed a series of SB vectors carrying either a DsRed or a human β-globin transgene that was encompassed by cHS4 insulator elements, and containing the SB100X transposase gene outside the SB-Tn unit within the same vector in cis configuration. These SB-Tn constructs were introduced into the K-562 erythroid cell line, and their presence in the genomes of host cells was analyzed by Southern blot analysis using non-radioactive probes. Many copies of SB-Tn insertions were detected in host cells regardless of transgene sequences or the presence of cHS4 insulator elements. Interestingly, the size difference of 2.4 kb between insulated SB and non-insulated controls did not reflect the proportional difference in copy numbers of inserted SB-Tns. We then attempted methylation-sensitive Southern blots to assess the potential influence of cHS4 insulator elements on the epigenetic modification of SB-Tn. Our results indicated that SB100X was able to integrate at multiple sites with the number of SB-Tn copies larger than 6 kb in size. In addition, the non-radioactive Southern blot protocols developed here will be useful to detect integrated SB-Tn copies in any mammalian cell type.

A universal system to select gene-modified hepatocytes in vivo.

Many genetic and acquired liver disorders are amenable to gene and/or cell therapy. However, the efficiencies of cell engraftment and stable genetic modification are low and often subtherapeutic. In particular, targeted gene modifications from homologous recombination are rare events. These obstacles could be overcome if hepatocytes that have undergone genetic modification were to be selectively amplified or expanded. We describe a universally applicable system for in vivo selection and expansion of gene-modified hepatocytes in any genetic background. In this system, the therapeutic transgene is coexpressed with a short hairpin RNA (shRNA) that confers modified hepatocytes with resistance to drug-induced toxicity. An shRNA against the tyrosine catabolic enzyme 4-OH-phenylpyruvate dioxygenase protected hepatocytes from 4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate, a small-molecule inhibitor of fumarylacetoacetate hydrolase. To select for specific gene targeting events, the protective shRNA was embedded in a microRNA and inserted into a recombinant adeno-associated viral vector designed to integrate site-specifically into the highly active albumin locus. After selection of the gene-targeted cells, transgene expression increased 10- to 1000-fold, reaching supraphysiological levels of human factor 9 protein (50,000 ng/ml) in mice. This drug resistance system can be used to achieve therapeutically relevant transgene levels in hepatocytes in any setting.

Regulating the expression of therapeutic transgenes by controlled intake of dietary essential amino acids

Widespread application of gene therapy will depend on the development of simple methods to regulate the expression of therapeutic genes. Here we harness an endogenous signaling pathway to regulate therapeutic gene expression through diet. The GCN2-eIF2α signaling pathway is specifically activated by deficiencies in any essential amino acid (EAA); EAA deficiency leads to rapid expression of genes regulated by ATF4-binding cis elements. We found that therapeutic genes under the control of optimized amino acid response elements (AAREs) had low basal expression and high induced expression. We applied our system to regulate the expression of TNFSF10 (TRAIL) in the context of glioma therapy and found that intermittent activation of this gene by EEA-deficient meals retained its therapeutic efficacy while abrogating its toxic effects on normal tissue. The GCN2-eIF2α pathway is expressed in many tissues, including the brain, and is highly specific to EAA deficiency. Our system may be particularly well suited for intermittent regulation of therapeutic transgenes over short or long time periods.

566. Targeted Lentiviral Vector Integration Mediated by the AAV Rep78 Protein

Our goal is to improve the safety of lentiviral vectors by shifting their integration preference to genomic “safe harbor” sites where therapeutic transgenes can integrate and function in a predictable manner. To do this, we have generated integrase-defective LV vectors (IDLVs) capable of inserting transgene sequences at predetermined genomic sites through homologous recombination (HR). To increase the frequency of HR, we employ genome editing approaches involving site-specific nucleases. A major drawback of this approach is safety concerns caused by inadvertent double strand breaks (DSBs) and non-homologous end-joining (NHEJ) events at unintended genomic (off target) sites. To bypass this risk, we are pursuing site-specific nucleases that do not involve DSBs as a potentially safer class of DNA-modifying agents for site-specific integration of transgenes in human cells. The sequence and strand-specific endonucleases Rep78 and Rep68 encoded by adeno-associated virus (AAV) have previously been reported to stimulate HR of plasmids bearing homology arms corresponding to the AAVS1 safe harbor locus on chromosome 19. We tested the integration efficiency of IDLVs containing short homology arms corresponding to the AAVS1 locus mediated by an engineered version of the AAV2-derived Rep78 protein in HEK293 cells. We compared the integration efficiency of IDLVs mediated by the Rep78 protein to that of an AAVS1-specific zinc finger nuclease (ZFN). Furthermore, we explored the potential of IDLV donor vectors for targeted integration mediated by Rep78 in human induced pluripotent stem cells (hiPSCs). The results obtained indicate that in the presence of the Rep78 protein, the integration efficiency of IDLVs was up to 8.3 fold higher compared to cells treated with the donor vector alone. Moreover, the efficiency of site-specific targeting mediated by Rep78 was higher than that of the AAVS1-specific ZFN. We also showed that Rep78 can successfully direct integration of IDLVs at the AAVS1 locus in hiPSCs. We anticipate that the approach used may ultimately contribute to the reduction of risks associated with LV vectors.

413. Effect of Adenoviral Death Protein on NIS-Based Iodine Therapy and Imaging for Pancreatic Cancer

In this study, we assess our novel Oncolytic Adenovirus (OAd) expressing a dual function therapeutic and imaging transgene, the sodium-iodide symporter (NIS) as a promising alternative for pancreatic cancer treatment. We hypothesized that NIS expression in pancreatic cancer will induce uptake of radioiodine and allow noninvasive SPECT/CT imaging with 123I. We designed our OAd-NIS to overcome the low efficacy of previous vectors. These viruses have a modified Ad5/Ad3 fiber-knob shown to improve the poor transduction of pancreatic cancer cells. Viral replication is controlled under the Cox2 promoter allowing specific delivery of viral genes, and most of adenoviral E3 genes are deleted and replaced with NIS and Adenovirus Death Protein (ADP) genes. We have previously demonstrated significantly improved ability of ADP-overexpressing OAds to enhance viral release and cytolytic activity. Although improved oncolysis enhances viral release and improve the therapeutic effect in solid tumors, it can potentially reduce radioiodide tracer uptake. To test this possibility we designed and compared identical vectors with ADP (OAd-NIS-ADP) and without ADP (OAd-NIS-noADP) in vitro and in vivo. We first compared virus killing ability in pancreatic cancer cell lines. The ADP+ vector was significantly more cytolytic that no-ADP counterpart and showed improved viral replication and spread. Cox2-controlled OAd did not produce cell death in Cox2-negative control, confirming selectivity. We next assessed OAds ability to induce NIS protein expression and radioiodine uptake. NIS-OAds efficiently produce glycosylated NIS multimers as early as 2-days post infection. Infection with no-ADP vectors resulted in a significantly greater radioiodine uptake (125I) compared to ADP+ viruses. Importantly, ex vivo uptake test in human patient tissues confirmed the high level of NIS in pancreatic adenocarcinoma samples, and revealed no 125I uptake in normal pancreas. We further assessed the ability of our vectors to visualize human pancreatic cancer xenografts in a mouse model by monitoring 99mTcO4− accumulation with SPECT-CT. The OAd-NIS-noADP showed an earlier and more sustained radioisotope uptake when compared to ADP+ supporting its use as a more sensitive diagnostic tool for pancreatic cancer. Of contrary, the ADP+ vector significantly outperformed OAd-NIS-noADP in tumor shrinkage. These results suggest that while OAd-NIS-noADP produces a greater radiotracer uptake and can be used as a diagnostic tool, its ADP+ counterpart results in a better therapeutic effect when applied as a monotherapy. It is now essential to estimate the therapeutic ability of both vectors upon combination with I-131 (ongoing study). Further investigation will include the imaging and therapeutic studies in patient-derived xenografts. Ultimately, the goal of our research is to design a multimodal therapy with radiation and oncolytic virus for diagnosis and therapy of cancers.

485. ZFN-Mediated Liver-Targeting Gene Therapy Corrects Systemic and Neurological Disease of Mucopolysaccharidosis Type I

Mucopolysaccharidosis type I (MPS I) is characterized by progressive neurodegeneration, and premature death (<10 years). Caused by α-L-iduronidase (IDUA) deficiency and systemic accumulation of glycosaminoglycans (GAG), current therapies include stem cell transplant (with significant risk of morbidity and mortality), and enzyme replacement therapy (requiring costly and frequent long therapeutic infusion sessions). A promising alternative is genome editing by integration of a therapeutic hIDUA transgene using zinc finger nucleases (ZFN). We have previously demonstrated AAV-mediated in vivo targeting of the albumin locus as a “safe harbor” for coagulation factors, correcting clotting defects in hemophilic mice. Targeted insertion of a transgene into the genome offers multiple advantages. We can exploit the highly active albumin enhancer/promoter, such that stably modified hepatocytes demonstrate long-term transgene expression. Also, utilization of an endogenous promoter allows efficient packaging of the transgene donor into AAV. MPS I mice (n=8 per gender, 4-9 weeks old) were injected with a single dose of AAV2/8 encoding albumin-targeted ZFN and a donor encoding a partial hIDUA cDNA. MiSeq analysis showed that treated mice displayed significant levels of insertions/deletions (indels) (up to 56%) at the target locus, demonstrating efficient delivery and expression of the albumin ZFNs. Significant plasma IDUA activity was also observed in the ZFN+hIDUA donor treated mice, up to 10-fold of wild type levels, throughout the 120-day study. Urine GAG levels serving as a biomarker for hIDUA activity were reduced significantly (up to 90%). IDUA levels in these animals increased significantly in liver (up to 14 fold), heart, lung, muscle and spleen. Tissue GAG levels were significantly reduced in liver (by 91%), heart (85%), lung (86%), muscle (68%) and spleen (84%). Barnes maze tests at the end of the study showed that ZFN+hIDUA donor treated MPS I mice achieved significant neurological benefits compared with untreated MPS I mice.ZFN-mediated genome editing of hepatocytes in vivo thus resulted in high and stable levels of hIDUA expression in treated animals. This enzyme was secreted into plasma and then taken up by secondary tissues, leading to significant GAG reduction. The correction of the observable neurological impairment in MPS I mice suggests that a small but sufficient amount of hIDUA protein crossed the blood-brain barrier in these animals.The study provides a general “proof-of-concept” for treatment of lysosomal diseases.

684. Enhanced FVIII AAV Vector Cassette Produces Improved Virus Yields and Supraphysiological FVIII Levels In Vivo

Hemophilia A is the most common inherited bleeding disorder in humans and is caused by a deficiency of blood coagulation factor VIII (FVIII). This disease is an ideal candidate for liver-directed gene therapy, as even modest increases in FVIII activity (>1% of normal) can ameliorate the severe bleeding phenotype. Adeno-associated viral (AAV) vectors have shown great promise in both preclinical and clinical trials to efficiently deliver therapeutic transgenes to the liver. Suboptimal virus yields of AAV comprising human FVIII (hFVIII) for use in human therapy have hampered clinical scale manufacturing, both from mammalian (HEK293) and insect cells (Baculovirus system). We optimized an hFVIII AAV vector cassette to improve both virus yields and liver-specific hFVIII expression. Compared to historical hFVIII AAV vector cassettes that routinely produce 20% of standard, non-hFVIII containing AAV2/6 virus preparations, the improved hFVIII AAV vector cassette produced yields 100% of standard AAV2/6, both from HEK293 cells at the cell factory (CF) scale, but also in the Baculovirus system at large scale. Improved yields were also observed using other AAV serotypes including hFVIII AAV2/8 and AAV2/9. Using virus produced from the improved hFVIII AAV cassette we transduced mice at 6E+10 vg/mouse (~2E+12 vg/kg). Peak levels of hFVIII protein were achieved in mouse plasma at day 14 and represented supraphysiological levels of normal hFVIII in humans with serotypes AAV2/8 and AAV2/9; up to 337% and 516% of normal hFVIII plasma levels respectively (1U = 200 ng/mL = 100%). Transducing mice with serotype AAV2/6, known to be inefficient at transducing mouse liver, at 6E+10 vg/mouse (~2E+12 vg/kg) we achieved a mean peak value of 91.9 % +/- 15.5 SEM (n=6) of normal hFVIII plasma levels in humans. At a higher dose representing ~6E+12 vg/kg we achieved a mean peak value of hFVIII plasma over six independent in vivo mouse studies of 169.2 % +/- 10.1 SEM (n = 36). Supraphysiological hFVIII plasma levels were also achieved in non-human primates at a dose of 2E+12 vg/kg using serotype AAV2/6, representing up to 840% of normal hFVIII levels (8.4U). The robust production of hFVIII from the enhanced hFVIII AAV vector could significantly reduce the dose required to achieve therapeutic levels in human subjects. Additionally the improved hFVIII AAV vector cassette will enable clinical scale manufacturing.

692. Genome Editing for Personalized Gene Therapy of IVSI-110 Beta-Thalassemia

Thalassemia is amongst the commonest single-gene disorders worldwide, caused by deficient production of α- or β-globin. Of particular clinical relevance is β-thalassemia, which as a severe monogenic disease of the hematopoietic system is an ideal target for gene therapy, either by gene addition or gene correction. Problems inherent to the gene-augmentation approach for β-thalassemia, including insertional mutagenesis or low expression of the therapeutic transgene, may be avoided using targeted gene correction of mutations with site-specific designer nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 or transcription activator-like effector nucleases (TALENs). Our study is focused on the development of a personalized gene-correction therapy of the common β-thalassemia mutation, HBBIVS1-110, which in most Mediterranean and many Western countries has a frequency of above 20% (with 80% on the island of Cyprus) amongst β-thalassemia carriers. HBBIVS1-110 creates an abnormal splice-acceptor site in intron I of the β-globin gene, leading to a pre-mature in-frame stop codon in the aberrantly spliced mRNA and to early transfusion dependence of homozygotes. We have developed and evaluated designer nucleases targeting the site of the HBBIVS1-110 β-thalassemia mutation, including direct comparison of targeted disruption efficiency and toxicity for CRISPR/Cas9 and TALEN tools in human embryonic kidney cells and in murine erythroleukemia (MEL) cells carrying a HBBIVS1-110mutant transgene based on the GLOBE (MA821) vector. Using the latter, we have assessed the therapeutic efficiency and cleavage properties of these designer nucleases. Analyses included immunoblots, absolute quantification of correctly and aberrantly spliced HBB mRNAs using multiplex RT-qPCR, sequencing and characterization of the resulting genome-editing events. We noted superior performance of several TALEN pairs compared to a single CRISPR/Cas9 nuclease suitable for the target site. Moreover, we demonstrate a significant increase of correct splicing and β-globin chain synthesis for genome-edited transgenic MEL MA821 HBBIVS1-110 cells. For the two most efficient TALEN pairs, β-globin protein levels in pools of edited cells reach over 20% of that detected in MEL cells harbouring a wild-type MA821 HBB control, up from 1% for the mock-treated MA821 HBBIVS1-110 control. Our findings validate our approach and indicate its suitability also for the correction of other intronic disease-causing mutations.