Erik R. Olson

Sleeping Beauty‐mediated correction of Fanconi anemia type C

The Sleeping Beauty (SB) transposon system can insert defined sequences into chromosomes to direct the extended expression of therapeutic genes. Our goal is to develop the SB system for nonviral complementation of Fanconi anemia (FA), a rare autosomal recessive disorder accompanied by progressive bone marrow failure.

Simple and Efficient Methods for Enrichment and Isolation of Endonuclease Modified Cells

The advent of Transcription Activator-Like Effector Nucleases (TALENs), and similar technologies such as CRISPR, provide a straightforward and cost effective option for targeted gene knockout (KO). Yet, there is still a need for methods that allow for enrichment and isolation of modified cells for genetic studies and therapeutics based on gene modified human cells. We have developed and validated two methods for simple enrichment and isolation of single or multiplex gene KOs in transformed, immortalized, and human progenitor cells. These methods rely on selection of a phenotypic change such as resistance to a particular drug or ability to grow in a selective environment. The first method, termed co-transposition, utilizes integration of a piggyBac transposon vector encoding a drug resistance gene. The second method, termed co-targeting, utilizes TALENs to KO any gene that when lost induces a selectable phenotype. Using these methods we also show removal of entire genes and demonstrate that TALENs function in human CD34+ progenitor cells. Further, co-transposition can be used to generate conditional KO cell lines utilizing an inducible cDNA rescue transposon vector. These methods allow for robust enrichment and isolation of KO cells in a rapid and efficient manner.

Transgene Expression in Dogs After Liver-Directed Hydrodynamic Delivery of Sleeping Beauty Transposons Using Balloon Catheters

The Sleeping Beauty transposon system has been extensively tested for integration of reporter and therapeutic genes in vitro and in vivo in mice. Dogs were used as a large animal model for human therapy and minimally invasive infusion of DNA solutions. DNA solutions were delivered into the entire liver or the left side of the liver using balloon catheters for temporary occlusion of venous outflow. A peak intravascular pressure between 80 and 140 mmHg supported sufficient DNA delivery in dog liver for detection of secretable reporter proteins. Secretable reporters allowed monitoring of the time course of gene products detectable in the circulation postinfusion. Canine secreted alkaline phosphatase reporter protein levels were measured in plasma, with expression detectable for up to 6 weeks, while expression of canine erythropoietin was detectable for 7-10 days. All animals exhibited a transient increase in blood transaminases that normalized within 10 days; otherwise the treated animals were clinically normal. These results demonstrate the utility of a secreted reporter protein for real-time monitoring of gene expression in the liver in a large animal model but highlight the need for improved delivery in target tissues to support integration and long-term expression of Sleeping Beauty transposons.

Role of transgene regulation in ex vivo lentiviral correction of artemis deficiency

Artemis is a single-stranded endonuclease, deficiency of which results in a radiation-sensitive form of severe combined immunodeficiency (SCID-A) most effectively treated by allogeneic hematopoietic stem cell (HSC) transplantation and potentially treatable by administration of genetically corrected autologous HSCs. We previously reported cytotoxicity associated with Artemis overexpression and subsequently characterized the human Artemis promoter with the intention to provide Artemis expression that is nontoxic yet sufficient to support immunodevelopment. Here we compare the human Artemis promoter (APro) with the moderate-strength human phosphoglycerate kinase (PGK) promoter and the strong human elongation factor-1α (EF1α) promoter to regulate expression of Artemis after ex vivo lentiviral transduction of HSCs in a murine model of SCID-A. Recipient animals treated with the PGK-Artemis vector exhibited moderate repopulation of their immune compartment, yet demonstrated a defective proliferative T lymphocyte response to in vitro antigen stimulation. Animals treated with the EF1α-Artemis vector displayed high levels of T lymphocytes but an absence of B lymphocytes and deficient lymphocyte function. In contrast, ex vivo transduction with the APro-Artemis vector supported effective immune reconstitution to wild-type levels, resulting in fully functional T and B lymphocyte responses. These results demonstrate the importance of regulated Artemis expression in immune reconstitution of Artemis-deficient SCID.

Prolonged Expression of Secreted Enzymes in Dogs After Liver-Directed Delivery of Sleeping Beauty Transposons: Implications for Non-Viral Gene Therapy of Systemic Disease

The non-viral, integrating Sleeping Beauty (SB) transposon system is efficient in treating systemic monogenic disease in mice, including hemophilia A and B caused by deficiency of blood clotting factors and mucopolysaccharidosis types I and VII caused by α-L-iduronidase (IDUA) and β-glucuronidase (GUSB) deficiency, respectively. Modified approaches of the hydrodynamics-based procedure to deliver transposons to the liver in dogs were recently reported. Using the transgenic canine reporter secreted alkaline phosphatase (cSEAP), transgenic protein in the plasma was demonstrated for up to 6 weeks post infusion. This study reports that immunosuppression of dogs with gadolinium chloride (GdCl3) prolonged the presence of cSEAP in the circulation up to 5.5 months after a single vector infusion. Transgene expression declined gradually but appeared to stabilize after about 2 months at approximately fourfold baseline level. Durability of transgenic protein expression in the plasma was inversely associated with transient increase of liver enzymes alanine transaminase and aspartate transaminase in response to the plasmid delivery procedure, which suggests a deleterious effect of hepatocellular toxicity on transgene expression. GdCl3 treatment was ineffective for repeat vector infusions. In parallel studies, dogs were infused with potentially therapeutic transposons. Activities of transgenic IDUA and GUSB in plasma peaked at 50-350% of wildtype, but in the absence of immunosuppression lasted only a few days. Transposition was detectable by excision assay only when the most efficient transposase, SB100X, was used. Dogs infused with transposons encoding canine clotting factor IX (cFIX) were treated with GdCl3 and showed expression profiles similar to those in cSEAP-infused dogs, with expression peaking at 40% wt (2 μg/mL). It is concluded that GdCl3 can support extended transgene expression after hydrodynamic introduction of SB transposons in dogs, but that alternative regimens will be required to achieve therapeutic levels of transgene products.

A Broad Range of Dose Optima Achieve High-level, Long-term Gene Expression After Hydrodynamic Delivery of Sleeping Beauty Transposons Using Hyperactive SB100x Transposase

The Sleeping Beauty (SB) transposon system has been shown to enable long-term gene expression by integrating new sequences into host cell chromosomes. We found that the recently reported SB100x hyperactive transposase conferred a surprisingly high level of long-term expression after hydrodynamic delivery of luciferase-encoding reporter transposons in the mouse. We conducted dose-ranging studies to determine the effect of varying the amount of SB100x transposase-encoding plasmid (pCMV-SB100x) at a set dose of luciferase transposon and of varying the amount of transposon-encoding DNA at a set dose of pCMV-SB100x in hydrodynamically injected mice. Animals were immunosuppressed using cyclophosphamide in order to prevent an antiluciferase immune response. At a set dose of transposon DNA (25 µg), we observed a broad range of pCMV-SB100x doses (0.1–2.5 µg) conferring optimal levels of long-term expression (>1011 photons/second/cm2). At a fixed dose of 0.5 μg of pCMV-SB100x, maximal long-term luciferase expression (>1010 photons/second/cm2) was achieved at a transposon dose of 5–125 μg. We also found that in the linear range of transposon doses (100 ng), co-delivering the CMV-SB100x sequence on the same plasmid was less effective in achieving long-term expression than delivery on separate plasmids. These results show marked flexibility in the doses of SB transposon plus pCMV-SB100x that achieve maximal SB-mediated gene transfer efficiency and long-term gene expression after hydrodynamic DNA delivery to mouse liver.

439. Prolonged Expression of Secreted Enzymes in Dogs After Liver Delivery of Sleeping Beauty Transposons: Implications for Non-Viral Gene Therapy of Mucopolysaccharidoses Types I and VII

The non-viral integrating vector the Sleeping Beauty (SB) transposon system is efficient in treating systemic monogenic diseases in mice including mucopolysaccharidosis (MPS) types I and VII caused by α-iduronidase (IDUA) and β-glucuronidase (GUSB) deficiency, respectively. More recently we have used modified approaches of the hydrodynamic procedure to deliver therapeutic transposons to dog liver. Reproducible delivery and transposition in dogs are about 1% the levels in mice. Using a transgenic canine reporter secreted alkaline phosphatase (cSEAP), in the absence of immune suppression we can detect transgenic protein for up to six weeks post infusion using catheter-mediated hydrodynamic delivery. A proof-of-principle immunomodulation using GdCl3 to block macrophages in liver and spleen prolonged the presence of the cSEAP protein in circulation from 6 weeks to up to at least 5 months after a single vector infusion. We achieved stabilized activity in one dog at about 2-fold of baseline values. Durability of cSEAP in serum was inversely correlated with transient increase of liver enzymes ALT and AST in response to the vector delivery procedure, pointing to the deleterious effect of hepatocellular toxicity on transgene maintenance. However, GdCl3 immunomodulation was ineffective for repeat vector infusions, suggesting a possibility of an alternative, more potent immunosuppression regimen. Evidence of transposition was obtained with the most efficient transposase SB100X but not with SB11. For transgenic IDUA and GUSB, therapeutic activity in serum peaked at 50-350% of wild-type at 2-4 days post-treatment, but lasted only a few days. The differences in levels and duration of detection of cSEAP in the blood compared to those of IDUA and GUSB may be in part due to the facilitated uptake of lysosomal enzymes into cells compared to cSEAP. Longer endurance of transgenic proteins at therapeutic levels may be possible in SB-treated dogs using alternative immunosuppressive regimens.

126. Non-Viral Gene Therapy By Liver-Directed Hydrodynamic Delivery of Sleeping Beauty Transposons to Treat Hemophilia and Mucopolysaccharidoses in Dogs

The goal of gene therapy is to achieve sustained expression of a transgene encoding an enzyme deficient in patients. Others and we have reported effectiveness of the Sleeping Beauty (SB) transposon system for gene therapy of both hemophilia B and mucopolysaccharidoses (MPS) types I and VII in adult mice. Although more than 99% of transgene expression comes from the liver following hydrodynamic delivery, restoration of deficient enzyme activity in other organs can be achieved through metabolic cross-correction whereby IDUA enters the circulation and is distributed to other tissues. However, the efficacy of hydrodynamic delivery to treat animals larger than mice has been discouraging. We have tested the use of balloon-catheters for intravascular infusion and expression of SB transposons in the liver of dogs as a large animal model for human therapy. Balloon-catheters were introduced under fluoroscopic guidance through either the jugular or the femoral vein and positioned in the inferior vena cava for occlusion of venous outflow of the and retrograde infusion through the left hepatic vein. Infusions were 10 seconds in duration and delivered volumes of DNA solution equal to about three times the estimated blood volume of the left side of the liver. Using canine secreted alkaline phosphatase (cSEAP) as a reporter we have developed effective protocols for infusion of transposons into canine liver as a scale-up for preclinical gene therapy studies in large animal models of human disease. We tested our protocol on both Factor IX (FIX)-deficient and beta-glucuronidase (GUSB)-deficient young dogs by hydrodynamically infusing two plasmids, one that harbors a T2-SB transposon containing either a canine FIX gene or a canine GUSB gene under transcriptional direction of a CAGGS or liver specific promoter (LSP) and a second carrying either the SB11 or SB100X transposase regulated by the CMV early promoter. Duration of FIX and GUSB expression in the treated dogs was transient, lasting only about 1 – 8 weeks. PCR analysis of plasmids in liver suggest that the efficiency of catheter-mediated delivery to canine livers is approximately 0.1-1% that in the mouse, which accounts for the relatively poor levels of transgene activity in the larger animals. We discuss our hypothesis that the larger vascular net with many more bifurcations in the livers of large animals lowers both impulse and shear forces necessary for effective hydrodynamic delivery in the liver.

379. Delivery of Human Clotting Factors By Expression from B lymphocytes Genetically Engineered Using the Sleeping Beauty Transposon System

Hemophilia A and hemophilia B are X-linked, genetic disorders that are caused by defective or deficient coagulation factor VIII and IX, respectively, resulting in the inability to form blood clots and sustained bleeding after trauma or injury. Recombinant clotting factor protein is currently used to treat hemophilia at a high cost per patient. As a therapeutic approach for hemophilia, gene transfer has the potential to provide more consistent levels of circulating clotting factor over an extended period of time for more cost-effective treatment. Moreover, only modest levels of FVIII or FIX expression (2%-5% of normal) can improve clinical outcomes. We are using the Sleeping Beauty (SB) transposon system to engineer autologous human B cells for secretion of clotting factors as a cellular therapy for hemophilia. An in vitro system for expansion and differentiation of memory B cells into plasma cells has been developed. Plasma cells are suitable for sustained delivery of FVIII or FIX, since they secrete high levels of protein and may survive for years in vivo. For human FIX expression, we assembled an SB transposon with the human FIX coding sequence (codon-optimized with R338L mutation for enhanced potency) regulated by the CAGS promoter. Elevated levels of hFIX in cultures of B lymphoblastoid cells required co-electroporation of the hFIX transposon along with an SB transposase encoding plasmid, demonstrating the role of transposition in achieving extended hFIX expression. There have been remarkable advances recently in the treatment of hemophilia B by systemic AAV8-hFIX administration, but similar treatment of hemophilia A presents a significant challenge due to the size of the FVIII-encoding sequence and the complexity of the protein. We previously demonstrated B-domain deleted hFVIII expression and correction of clotting dysfunction in FVIII deficient mice by hydrodynamic delivery using the SB transposon system (Ohlfest et al, Blood 105: 2691, 2005). Current studies are focused on identifying conditions for effective hFVIII transposon delivery and long term expression in primary human B cells after Sleeping Beauty-mediated transposition. Results from these studies will be applicable to the development of a clinical protocol for treatment of human hemophilia by infusion of B cells genetically engineered using the Sleeping Beauty transposon system.

Sleeping Beauty-Mediated Drug Resistance Gene Transfer in Human Hematopoietic Progenitor Cells

The Sleeping Beauty (SB) transposon system can insert sequences into mammalian chromosomes, supporting long-term expression of both reporter and therapeutic genes. Hematopoietic progenitor cells (HPCs) are an ideal therapeutic gene transfer target as they are used in therapy for a variety of hematologic and metabolic conditions. As successful SB-mediated gene transfer into human CD34(+) HPCs has been reported by several laboratories, we sought to extend these studies to the introduction of a therapeutic gene conferring resistance to methotrexate (MTX), potentially providing a chemoprotective effect after engraftment. SB-mediated transposition of hematopoietic progenitors, using a transposon encoding an L22Y variant dihydrofolate reductase fused to green fluorescent protein, conferred resistance to methotrexate and dipyridamole, a nucleoside transport inhibitor that tightens MTX selection conditions, as assessed by in vitro hematopoietic colony formation. Transposition of individual transgenes was confirmed by sequence analysis of transposon-chromosome junctions recovered by linear amplification-mediated PCR. These studies demonstrate the potential of SB-mediated transposition of HPCs for expression of drug resistance genes for selective and chemoprotective applications.