Lysosomal storage disease: gene therapy on both sides of the blood-brain barrier.

Most lysosomal storage disorders affect the nervous system as well as other tissues and organs of the body. Previously, the complexities of these diseases, particularly in treating neurologic abnormalities, were too great to surmount. However, based on recent developments there are realistic expectations that effective therapies are coming soon. Gene therapy offers the possibility of affordable, comprehensive treatment associated with these diseases currently not provided by standards of care. With a focus on correction of neurologic disease by systemic gene therapy of mucopolysaccharidoses types I and IIIA, we review some of the major recent advances in viral and non-viral vectors, methods of their delivery and strategies leading to correction of both the nervous and somatic tissues as well as evaluation of functional correction of neurologic manifestations in animal models. We discuss two questions: what systemic gene therapy strategies work best for correction of both somatic and neurologic abnormalities in a lysosomal storage disorder and is there evidence that targeting peripheral tissues (e.g., in the liver) has a future for ameliorating neurologic disease in patients?

The Sleeping Beauty transposon system: a non-viral vector for gene therapy.

Over the past decade, the Sleeping Beauty (SB) transposon system has been developed as the leading non-viral vector for gene therapy. This vector combines the advantages of viruses and naked DNA. Here we review progress over the last 2 years in vector design, methods of delivery and safety that have supported its use in the clinic. Currently, the SB vector has been validated for ex vivo gene delivery to stem cells, including T-cells for the treatment of lymphoma. Progress in delivery of SB transposons to liver for treatment of various systemic diseases, such as hemophilia and mucopolysaccharidoses types I and VII, has encountered some problems, but even here progress is being made.

Lysosomal enzyme can bypass the blood-brain barrier and reach the CNS following intranasal administration.

Here we provide the first evidence that therapeutic levels of a lysosomal enzyme can bypass the blood-brain barrier following intranasal administration. α-L-iduronidase (IDUA) activity was detected throughout the brains of IDUA-deficient mice following a single intranasal treatment with concentrated Aldurazyme® (laronidase) and was also detected after intranasal treatment with an adeno-associated virus (AAV) vector expressing human IDUA. These results suggest that intranasal routes of delivery may be efficacious in the treatment of lysosomal storage disorders.

Sleeping Beauty-mediated correction of Fanconi anemia type C.

BACKGROUND: 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. METHODS: We used a CytoPulse electroporation system (CytoPulse, Glen Burnie, MD, USA) to introduce SB transposons into human lymphoblastoid cells (LCL) derived from both Fanconi anemia type C (FA-C) defective and normal patients. Correction of the FA-C defect was assessed by resistance to mitomycin C, a DNA-crosslinking agent. RESULTS: Culture of both cell types with the antioxidant N-acetyl- l-cysteine improved cell viability after electroporation. Co-delivery of enhanced green fluorescent protein (GFP) transposon with SB100X transposase-encoding plasmid supported a 50- to 90-fold increase in stable GFP expression compared to that observed in the absence of SB100X for normal LCL, but in FA-C defective LCL SB100X enhancement of stable GFP-expression was a more moderate five- to 13-fold. SB-mediated integration and expression of the FA-C gene was demonstrated by the emergence of a mitomycin C-resistant population bearing characteristic transposon-chromosome junction sequences and exhibiting a mitomycin dose response identical to that of normal LCL. CONCLUSIONS: The SB transposon system achieved stable expression of therapeutic FA-C genes, complementing the genetic defect in patient-derived cells by nonviral gene transfer.

Duration of expression and activity of Sleeping Beauty transposase in mouse liver following hydrodynamic DNA delivery.

The Sleeping Beauty (SB) transposon system can direct integration of DNA sequences into mammalian genomes. The SB system comprises a transposon and transposase that "cuts" the transposon from a plasmid and "pastes" it into a recipient genome. The transposase gene may integrate very rarely and randomly into genomes, which has led to concerns that continued expression might support continued remobilization of transposons and genomic instability. Consequently, we measured the duration of SB11 transposase expression needed for remobilization to determine whether continued expression might be a problem. The SB11 gene was expressed from the plasmid pT2/mCAGGS-Luc//UbC-SB11 that contained a luciferase expression cassette in a hyperactive SB transposon. Mice were imaged and killed at periodic intervals out to 24 weeks. Over the first 2 weeks, the number of plasmids with SB11 genes and SB11 mRNA dropped about 90 and 99.9%, respectively. Expression of the luciferase reporter gene in the transposon declined about 99% and stabilized for 5 months at nearly 1,000-fold above background. In stark contrast, transposition-supporting levels of SB11 mRNA lasted only about 4 days postinfusion. Thus, within the limits of current technology, we show that SB transposons appear to be as stably integrated as their viral counterparts.

Systemic correction of storage disease in MPS I NOD/SCID mice using the sleeping beauty transposon system.

The Sleeping Beauty (SB) transposon system is a nonviral vector that directs transgene integration into vertebrate genomes. We hydrodynamically delivered SB transposon plasmids encoding human alpha-L-iduronidase (hIDUA) at two DNA doses, with and without an SB transposase gene, to NOD.129(B6)-Prkdc(scid) IDUA(tm1Clk)/J mice. In transposon-treated, nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with mucopolysaccharidosis type I (MPS I), plasma IDUA persisted for 18 weeks at levels up to several hundred-fold wild-type (WT) activity, depending on DNA dose and gender. IDUA activity was present in all examined somatic organs, as well as in the brain, and correlated with both glycosaminoglycan (GAG) reduction in these organs and level of expression in the liver, the target of transposon delivery. IDUA activity was higher in the treated males than in females. In females, omission of transposase source resulted in significantly lower IDUA levels and incomplete GAG reduction in some organs, confirming the positive effect of transposition on long-term IDUA expression and correction of the disease. The SB transposon system proved efficacious in correcting several clinical manifestations of MPS I in mice, including thickening of the zygomatic arch, hepatomegaly, and accumulation of foamy macrophages in bone marrow and synovium, implying potential effectiveness of this approach in treatment of human MPS I.

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.

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 ß-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.

NMR structural analysis of Sleeping Beauty transposase binding to DNA.

The Sleeping Beauty (SB) transposon is the most widely used DNA transposon in genetic applications and is the only DNA transposon thus far in clinical trials for human gene therapy. In the absence of atomic level structural information, the development of SB transposon relied primarily on the biochemical and genetic homology data. While these studies were successful and have yielded hyperactive transposases, structural information is needed to gain a mechanistic understanding of transposase activity and guides to further improvement. We have initiated a structural study of SB transposase using Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) spectroscopy to investigate the properties of the DNA-binding domain of SB transposase in solution. We show that at physiologic salt concentrations, the SB DNA-binding domain remains mostly unstructured but its N-terminal PAI subdomain forms a compact, three-helical structure with a helix-turn-helix motif at higher concentrations of NaCl. Furthermore, we show that the full-length SB DNA-binding domain associates differently with inner and outer binding sites of the transposon DNA. We also show that the PAI subdomain of SB DNA-binding domain has a dominant role in transposase's attachment to the inverted terminal repeats of the transposon DNA. Overall, our data validate several earlier predictions and provide new insights on how SB transposase recognizes transposon DNA.

Quantitative analysis of α-L-iduronidase expression in immunocompetent mice treated with the Sleeping Beauty transposon system.

The Sleeping Beauty transposon system, a non-viral, integrating vector that can deliver the alpha-L-iduronidase-encoding gene, is efficient in correcting mucopolysaccharidosis type I in NOD/SCID mice. However, in previous studies we failed to attain reliable long-term alpha-L-iduronidase expression in immunocompetent mice. Here, we focused on achieving sustained high-level expression in immunocompetent C57BL/6 mice. In our standard liver-directed treatment we hydrodynamically infuse mice with plasmids containing a SB transposon-encoding human alpha-L-iduronidase, along with a source of SB transposase. We sought to 1) minimize expression of the therapeutic enzyme in antigen-presenting cells, while avoiding promoter shutdown and gender bias, 2) increase transposition efficiency and 3) improve immunosuppression. By using a liver-specific promoter to drive IDUA expression, the SB100X hyperactive transposase and transient cyclophosphamide immunosuppression we achieved therapeutic-level (>100 wild-type) stabilized expression for 1 year in 50% of C57BL/6 mice. To gain insights into the causes of variability in transgene expression, we quantified the rates of alpha-L-iduronidase activity decay vis-a-vis transposition and transgene maintenance using the data obtained in this and previous studies. Our analyses showed that immune responses are the most important variable to control in order to prevent loss of transgene expression. Cumulatively, our results allow transition to pre-clinical studies of SB-mediated alpha-L-iduronidase expression and correction of mucopolysaccharidosis type I in animal models.

Efficacy and safety of Sleeping Beauty transposon-mediated gene transfer in preclinical animal studies.

Sleeping Beauty (SB) transposons have been effective in delivering therapeutic genes to treat certain diseases in mice. Hydrodynamic gene delivery of integrating transposons to 5-20% of the hepatocytes in a mouse results in persistent elevated expression of the therapeutic polypeptides that can be secreted into the blood for activity throughout the animal. An alternative route of delivery is ex vivo transformation with SB transposons of hematopoietic cells, which then can be reintroduced into the animal for treatment of cancer. We discuss issues associated with the scale-up of hydrodynamic delivery to the liver of larger animals as well as ex vivo delivery. Based on our and others' experience with inefficient delivery to larger animals, we hypothesize that impulse, rather than pressure, is a critical determinant of the effectiveness of hydrodynamic delivery. Accordingly, we propose some alterations in delivery strategies that may yield efficacious levels of gene delivery in dogs and swine that will be applicable to humans. To ready hydrodynamic delivery for human application we address a second issue facing transposons used for gene delivery regarding their potential to "re-hop" from one site to another and thereby destabilize the genome. The ability to correct genetic diseases through the infusion of DNA plasmids remains an appealing goal.

Preferential delivery of the Sleeping Beauty transposon system to livers of mice by hydrodynamic injection.

Nonviral, DNA-mediated gene transfer is an alternative to viral delivery systems for expressing new genes in cells and tissues. The Sleeping Beauty (SB) transposon system combines the advantages of viruses and naked DNA molecules for gene therapy purposes; however, efficacious delivery of DNA molecules to animal tissues can still be problematic. Here we describe the hydrodynamic delivery procedure for the SB transposon system that allows efficient delivery to the liver in the mouse. The procedure involves rapid, high-pressure injection of a DNA solution into the tail vein. The overall procedure takes <1 h although the delivery into one mouse requires only a few seconds. Successful injections result in expression of the transgene in 5-40% of hepatocytes 1 d after injection. Several weeks after injection, transgene expression stabilizes at approximately 1% of the level at 24 h, presumably owing to integration of the transposons into chromosomes.

Prolonged expression of a lysosomal enzyme in mouse liver after Sleeping Beauty transposon-mediated gene delivery: implications for non-viral gene therapy of mucopolysaccharidoses.

BACKGROUND: The Sleeping Beauty (SB) transposon system is a non-viral vector system that can integrate precise sequences into chromosomes. We evaluated the SB transposon system as a tool for gene therapy of mucopolysaccharidosis (MPS) types I and VII. METHODS: We constructed SB transposon plasmids for high-level expression of human beta-glucuronidase (hGUSB) or alpha-L-iduronidase (hIDUA). Plasmids were delivered with and without SB transposase to mouse liver by rapid, high-volume tail-vein injection. We studied the duration of expressed therapeutic enzyme activity, transgene presence by PCR, lysosomal pathology by toluidine blue staining and cell-mediated immune response histologically and by immunohistochemical staining. RESULTS: Transgene frequency, distribution of transgene and enzyme expression in liver and the level of transgenic enzyme required for amelioration of lysosomal pathology were estimated in MPS I and VII mice. Without immunomodulation, initial GUSB and IDUA activities in plasma reached > 100-fold of wild-type (WT) levels but fell to background within 4 weeks post-injection. In immunomodulated transposon-treated MPS I mice plasma IDUA persisted for over 3 months at up to 100-fold WT activity in one-third of MPS I mice, which was sufficient to reverse lysosomal pathology in the liver and, partially, in distant organs. Histological and immunohistochemical examination of liver sections in IDUA transposon-treated WT mice revealed inflammation 10 days post-injection consisting predominantly of mononuclear cells, some of which were CD4- or CD8-positive. CONCLUSIONS: Our results demonstrate the feasibility of achieving prolonged expression of lysosomal enzymes in the liver and reversing MPS disease in adult mice with a single dose of therapeutic SB transposons.

Excision of Sleeping Beauty transposons: parameters and applications to gene therapy.

A major problem in gene therapy is the determination of the rates at which gene transfer has occurred. Our work has focused on applications of the Sleeping Beauty (SB) transposon system as a non-viral vector for gene therapy. Excision of a transposon from a donor molecule and its integration into a cellular chromosome are catalyzed by SB transposase. In this study, we used a plasmid-based excision assay to study the excision step of transposition. We used the excision assay to evaluate the importance of various sequences that border the sites of excision inside and outside the transposon in order to determine the most active sequences for transposition from a donor plasmid. These findings together with our previous results in transposase binding to the terminal repeats suggest that the sequences in the transposon-junction of SB are involved in steps subsequent to DNA binding but before excision, and that they may have a role in transposase-transposon interaction. We found that SB transposons leave characteristically different footprints at excision sites in different cell types, suggesting that alternative repair machineries operate in concert with transposition. Most importantly, we found that the rates of excision correlate with the rates of transposition. We used this finding to assess transposition in livers of mice that were injected with the SB transposon and transposase. The excision assay appears to be a relatively quick and easy method to optimize protocols for delivery of genes in SB transposons to mammalian chromosomes in living animals.