Elena L. Aronovich

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.

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

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.

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 ∼1% of the level at 24 h, presumably owing to integration of the transposons into chromosomes.

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.

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. Copyright

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?

Genotype-phenotype correspondence in Sanfilippo syndrome type B.

Sanfilippo syndrome type B, or mucopolysaccharidosis type IIIB, results from defects in the gene for alpha-N-acetylglucosaminidase (NAGLU); only a few mutations have been described. To rapidly identify most NAGLU mutations, an automated sequencing procedure was developed for analysis of the entire coding region, including exon-intron borders. By this method, eight affected families were studied, and the mutations in all 16 alleles were identified, more than doubling the number of published mutations for this gene. Eight mutations were described for the first time: five missense mutations (Y140C, Y455C, P521L, S612G, and R674C), two nonsense mutations (W675X and Q706X), and one 24-nucleotide insertion. Currently, 36% of all point mutations (8 of 22 alleles) involve R674, a codon having a CpG dinucleotide in the critical initial position. Other mutations were found in more than one family, raising the possibility that some may be relatively common and, possibly, ancient mutations. Six new nonpathological mutations were also identified and likely represent polymorphic variants of the NAGLU gene, two of which might alter enzyme level. Establishing genotype-phenotype relationships will be vital in the evaluation of experimental treatments such as gene therapy.

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

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.

Retroviral vector design studies toward hematopoietic stem cell gene therapy for mucopolysaccharidosis type I

To optimize a gene transfer system for hematopoietic stem cell gene therapy of patients with mucopolysaccharidosis (MPS) type I, 10 retroviral vectors were constructed to express the human α-L-iduronidase (IDUA) cDNA. These vectors were designed to evaluate the potential effects of specific promoters, the addition of selectable markers, and the use of multiple promoters versus an internal ribosome entry site for expression of IDUA and selectable maker genes. The effect of vector design was investigated in primary patient fibroblasts (FMPS) or murine fibroblast cell lines; while overall comparison of transgene expression was determined in patients’ peripheral blood lymphocytes (PBLMPS) and CD34+ progenitors (PBPCMPS). We observed that the human PGK promoter introduced the highest IDUA activity per 1% relative transgene frequency in FMPS. Use of the same promoter to separately regulate both the therapeutic gene and a drug-resistance gene resulted in decreased expression of the unselected gene. Co-selection using bicistronic vectors not only increased the number of transductants, but also elevated transgene expression under selective pressure in transgene-positive progenitors. Bicistronic vector LP1CD overcame down-regulation and practically introduced the highest IDUA level in unselected PBLMPS and an intermediate level in PBPCMPS. These studies provide a better understanding of factors contributing to efficient gene expression in hematopoietic cells.