Stephen R. Yant

Sarcoma derived from cultured mesenchymal stem cells

To study the biodistribution of MSCs, we labeled adult murine C57BL/6 MSCs with firefly luciferase and DsRed2 fluorescent protein using nonviral Sleeping Beauty transposons and coinfused labeled MSCs with bone marrow into irradiated allogeneic recipients. Using in vivo whole‐body imaging, luciferase signals were shown to be increased between weeks 3 and 12. Unexpectedly, some mice with the highest luciferase signals died and all surviving mice developed foci of sarcoma in their lungs. Two mice also developed sarcomas in their extremities. Common cytogenetic abnormalities were identified in tumor cells isolated from different animals. Original MSC cultures not labeled with transposons, as well as independently isolated cultured MSCs, were found to be cytogenetically abnormal. Moreover, primary MSCs derived from the bone marrow of both BALB/c and C57BL/6 mice showed cytogenetic aberrations after several passages in vitro, showing that transformation was not a strain‐specific nor rare event. Clonal evolution was observed in vivo, suggesting that the critical transformation event(s) occurred before infusion. Mapping of the transposition insertion sites did not identify an obvious transposon‐related genetic abnormality, and p53 was not overexpressed. Infusion of MSC‐derived sarcoma cells resulted in malignant lesions in secondary recipients. This new sarcoma cell line, S1, is unique in having a cytogenetic profile similar to human sarcoma and contains bioluminescent and fluorescent genes, making it useful for investigations of cellular biodistribution and tumor response to therapy in vivo. More importantly, our study indicates that sarcoma can evolve from MSC cultures.

Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system

The development of non-viral gene-transfer technologies that can support stable chromosomal integration and persistent gene expression in vivo is desirable. Here we describe the successful use of transposon technology for the nonhomologous insertion of foreign genes into the genomes of adult mammals using naked DNA. We show that the Sleeping Beauty transposase can efficiently insert transposon DNA into the mouse genome in approximately 5–6% of transfected mouse liver cells. Chromosomal transposition resulted in long-term expression (>5 months) of human blood coagulation factor IX at levels that were therapeutic in a mouse model of haemophilia B. Our results establish DNA-mediated transposition as a new genetic tool for mammals, and provide new strategies to improve existing non-viral and viral vectors for human gene therapy applications.

Extrachromosomal Recombinant Adeno-Associated Virus Vector Genomes Are Primarily Responsible for Stable Liver Transduction In Vivo

ABSTRACT Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Although the vector genomes are found both as extrachromosomes and as chromosomally integrated forms in hepatocytes, the relative proportion of each has not yet been clearly established. Using an in vivo assay based on the induction of hepatocellular regeneration via a surgical two-thirds partial hepatectomy, we have determined the proportion of integrated and extrachromosomal rAAV genomes in mouse livers and their relative contribution to stable gene expression in vivo. Plasma human coagulation factor IX (hF.IX) levels in mice originating from a chromosomally integrated hF.IX-expressing transposon vector remained unchanged with hepatectomy. This was in sharp contrast to what was observed when a surgical partial hepatectomy was performed in mice 6 weeks to 12 months after portal vein injection of a series of hF.IX-expressing rAAV vectors. At doses of 2.4 × 1011to 3.0 × 1011 vector genomes per mouse (n = 12), hF.IX levels and the average number of stably transduced vector genomes per cell decreased by 92 and 86%, respectively, after hepatectomy. In a separate study, one of three mice injected with a higher dose of rAAV had a higher proportion (67%) of integrated genomes, the significance of which is not known. Nevertheless, in general, these results indicate that, in most cases, no more than ∼10% of stably transduced genomes integrated into host chromosomes in vivo. Additionally, the results demonstrate that extrachromosomal, not integrated, genomes are the major form of rAAV in the liver and are the primary source of rAAV-mediated gene expression. This small fraction of integrated genomes greatly decreases the potential risk of vector-related insertional mutagenesis associated with all integrating vectors but also raises uncertainties as to whether rAAV-mediated hepatic gene expression can persist lifelong after a single vector administration.

The 37/67-Kilodalton Laminin Receptor Is a Receptor for Adeno-Associated Virus Serotypes 8, 2, 3, and 9

ABSTRACT Adeno-associated virus serotype 8 (AAV8) is currently emerging as a powerful gene transfer vector, owing to its capability to efficiently transduce many different tissues in vivo. While this is believed to be in part due to its ability to uncoat more readily than other AAV serotypes such as AAV2, understanding all the processes behind AAV8 transduction is important for its application and optimal use in human gene therapy. Here, we provide the first report of a cellular receptor for AAV8, the 37/67-kDa laminin receptor (LamR). We document binding of LamR to AAV8 capsid proteins and intact virions in vitro and demonstrate its contribution to AAV8 transduction of cultured cells and mouse liver in vivo. We also show that LamR plays a role in transduction by three other closely related serotypes (AAV2, -3, and -9). Sequence and deletion analysis allowed us to map LamR binding to two protein subdomains predicted to be exposed on the AAV capsid exterior. Use of LamR, which is constitutively expressed in many clinically relevant tissues and is overexpressed in numerous cancers, provides a molecular explanation for AAV8s broad tissue tropism. Along with its robust transduction efficiency, our findings support the continued development of AAV8-based vectors for clinical applications in humans, especially for tumor gene therapy.

High-resolution genome-wide mapping of transposon integration in mammals

ABSTRACT The Sleeping Beauty (SB) transposon is an emerging tool for transgenesis, gene discovery, and therapeutic gene delivery in mammals. Here we studied 1,336 SB insertions in primary and cultured mammalian cells in order to better understand its target site preferences. We report that, although widely distributed, SB integration recurrently targets certain genomic regions and shows a small but significant bias toward genes and their upstream regulatory sequences. Compared to those of most integrating viruses, however, the regional preferences associated with SB-mediated integration were much less pronounced and were not significantly influenced by transcriptional activity. Insertions were also distinctly nonrandom with respect to intergenic sequences, including a strong bias toward microsatellite repeats, which are predominantly enriched in noncoding DNA. Although we detected a consensus sequence consistent with a twofold dyad symmetry at the target site, the most widely used sites did not match this consensus. In conjunction with an observed SB integration preference for bent DNA, these results suggest that physical properties may be the major determining factor in SB target site selection. These findings provide basic insights into the transposition process and reveal important distinctions between transposon- and virus-based integrating vectors.

Transposition from a gutless adeno-transposon vector stabilizes transgene expression in vivo

A major limitation of adenovirus-mediated gene therapy for inherited diseases is the instability of transgene expression in vivo, which originates at least in part from the loss of the linear, extrachromosomal vector genomes. Herein we describe the production of a gene-deleted adenovirus–transposon vector that stably maintains virus-encoded transgenes in vivo through integration into host cell chromosomes. This system utilizes a donor transposon vector that undergoes Flp-mediated recombination and excision of its therapeutic payload in the presence of the Flp and Sleeping Beauty recombinases. Systemic in vivo delivery of this system resulted in efficient generation of transposon circles and stable transposase-mediated integration in mouse liver. Somatic integration was sufficient to maintain therapeutic levels of human coagulation Factor IX for more than six months in mice undergoing extensive liver proliferation. These vectors combine the versatility of adenoviral vectors with the integration capabilities of a eukaryotic DNA transposon and should prove useful in the treatment of genetic diseases.

Mutational Analysis of the N-Terminal DNA-Binding Domain of Sleeping Beauty Transposase: Critical Residues for DNA Binding and Hyperactivity in Mammalian Cells

ABSTRACT The N-terminal domain of the Sleeping Beauty (SB) transposase mediates transposon DNA binding, subunit multimerization, and nuclear translocation in vertebrate cells. For this report, we studied the relative contributions of 95 different residues within this multifunctional domain by large-scale mutational analysis. We found that each of four amino acids (leucine 25, arginine 36, isoleucine 42, and glycine 59) contributes to DNA binding in the context of the N-terminal 123 amino acids of SB transposase, as indicated by electrophoretic mobility shift analysis, and to functional activity of the full-length transposase, as determined by a quantitative HeLa cell-based transposition assay. Moreover, we show that amino acid substitutions within either the putative oligomerization domain (L11A, L18A, L25A, and L32A) or the nuclear localization signal (K104A and R105A) severely impair its ability to mediate DNA transposition in mammalian cells. In contrast, each of 10 single amino acid changes within the bipartite DNA-binding domain is shown to greatly enhance SBs transpositional activity in mammalian cells. These hyperactive mutations functioned synergistically when combined and are shown to significantly improve transposase affinity for transposon end sequences. Finally, we show that enhanced DNA-binding activity results in improved cleavage kinetics, increased SB element mobilization from host cell chromosomes, and dramatically improved gene transfer capabilities of SB in vivo in mice. These studies provide important insights into vertebrate transposon biology and indicate that Sleeping Beauty can be readily improved for enhanced genetic research applications in mammals.

Helper-Independent sleeping beauty Transposon–Transposase vectors for efficient nonviral gene delivery and persistent gene expression in vivo

Transposon-based vectors represent promising new tools for chromosomal transgene insertion and establishment of persistent gene expression in vivo. Here, we report the development of helper-independent transposon-transposase (HITT) vectors, which contain on single plasmids (i) a Sleeping Beauty (SB) transposon containing the transgene and (ii) a SB transposase expression cassette. To obtain an optimal level of transposase expression from HITT vectors, we determined the relative strength of a panel of different promoters in mouse liver and used these promoters to drive transposase expression from injected HITT vectors carrying a human alpha(1)-antitrypsin (hAAT) expression cassette flanked by transposon inverted repeats. By correlating promoter strength with stabilized serum hAAT levels, a narrow expression window supporting high-level transposition in the liver was defined. Peak levels of long-term gene expression were obtained with promoters 30- to 40-fold less active than CMV in mouse liver, whereas reduced stable levels of hAAT were detected with both weaker and stronger promoters. Injected HITT vectors induced transposase-dependent insertion of transposon DNA into the genome of at least 5-6% of transfected hepatocytes, generating levels of persistent hAAT expression that were 2- to 4-fold higher than with an optimized two-plasmid approach. In addition, we show that HITT vectors carrying a human factor IX (hFIX)-containing transposon support (i) long-term hFIX expression in normal mice and (ii) partial phenotypic correction in a mouse model of hemophilia B. SB-based HITT vectors represent a major advance in the establishment of persistent transgene expression from nonviral gene delivery systems and should prove useful for gene transfer to tissues or cell types in which transfection efficiencies are low.

Site-directed transposon integration in human cells

The Sleeping Beauty (SB) transposon is a promising gene transfer vector that integrates nonspecifically into host cell genomes. Herein, we attempt to direct transposon integration into predetermined DNA sites by coupling a site-specific DNA-binding domain (DBD) to the SB transposase. We engineered fusion proteins comprised of a hyperactive SB transposase (HSB5) joined via a variable-length linker to either end of the polydactyl zinc-finger protein E2C, which binds a unique sequence on human chromosome 17. Although DBD linkage to the C-terminus of SB abolished activity in a human cell transposition assay, the N-terminal addition of the E2C or Gal4 DBD did not. Molecular analyses indicated that these DBD-SB fusion proteins retained DNA-binding specificity for their respective substrate molecules and were capable of mediating bona fide transposition reactions. We also characterized transposon integrations in the presence of the E2C-SB fusion protein to determine its potential to target predefined DNA sites. Our results indicate that fusion protein-mediated tethering can effectively redirect transposon insertion site selection in human cells, but suggest that stable docking of integration complexes may also partially interfere with the cut-and-paste mechanism. These findings illustrate the feasibility of directed transposon integration and highlight potential means for future development.

Distinct pathways of genomic progression to benign and malignant tumors of the liver.

We used several of the genetic lesions commonly associated with human liver tumors to reconstruct genetic progression to hepatocellular carcinoma and adenoma in mouse models. We initiated tumorigenesis with a transgene of the protooncogene MET or by hydrodynamic transfection of MET in combination with other genes into the livers of adult animals. Hepatocellular carcinoma in both instances arose from cooperation between MET and constitutively active versions of β-catenin. In contrast, adenomas were produced by cooperation between MET and defective signaling through the transcription factor HNF1α. Prompted by these findings, we uncovered a coincidence between activation of the protein-tyrosine kinase encoded by MET and activating mutations of β-catenin in a subset of human hepatocellular carcinomas. Inactivation of MET transgenes led to regression of hepatocellular carcinomas despite the persistence of activated β-catenin. The tumors eventually recurred in the absence of MET expression, however, presumably after the occurrence of one or more events that cooperated with activated β-catenin in lieu of MET. These results offer insight into hepatic tumorigenesis, provide mouse models that should be useful in the further study of hepatic tumorigenesis and for preclinical testing, and identify a subset of human hepatocellular carcinomas that may be susceptible to combination therapy directed against Met and the Wnt signaling pathway.