Better positioned in stem cells

In this issue of Blood, Xue and colleagues describe stable gene transfer into human CD34⁺ hematopoietic stem-progenitor cells using an improved DNA transposon system, allowing transgene expression in multiple lineages of lympho-hematopoietic cells by in vitro and in vivo assays lasting for weeks and months.

Human hematopoietic stem-progenitor cells (HSPCs) enriched in CD34⁺ cells from cord blood (CB) and adult sources remain an attractive target for various gene therapy strategies. In the past decade, the efficiency of stable gene transfer and expression mediated by integrating retroviral vectors such as those from gamma-retroviruses and lentiviruses has improved significantly. However, limitations of integrating retroviral vectors have also become evident. An important safety issue is that these integrating vectors tend to insert at intragenic or promoter regions, resulting in aberrant activation of proto-oncogenes or silencing of growth-suppressor genes. Other drawbacks of using these recombinant gammaretroviral and lentiviral vectors include costly and complicated production (packaging) and purification processes, and requirement of cell activation before or during the viral infection.

Efforts to use naked DNA, such as plasmids, for gene transfer into HSPCs have been unsuccessful because they did not integrate into the cellular genome and were quickly diluted or lost in HSPCs. With the advent of DNA transposon systems a decade ago that have also since showed efficacy in human cell lines,¹  scientists have hoped that transposon-mediated transgene insertion would also be effective in human HSPCs. A transposon-mediated gene transfer and expression system consists of 2 separate plasmids. In the first plasmid, a transgene (and its regulatory elements) is flanked by 2 cis DNA elements required for transposition. The second plasmid encodes a specific DNA recombination enzyme called transposase. After delivery of both plasmids to mammalian cells (transfection), the transposase will cut and paste the transgene from the first plasmid into the host genome. The engineered transposon is only mobile (in or out of genomic DNA) when transposase is present. The first such engineered system is Sleeping Beauty (SB), a synthetic transposon of the Tc1/mariner superfamily that was resurrected from the salmon genome. Since then, several different transposon systems have been developed for mammalian cells. One is called PiggyBac (PB) first derived from the cabbage looper moth and recently tested in mammalian cells by several groups.^2-4  It is not surprising that PB is less efficient than the latest SB systems. Progressive improvements of the SB transposase for mammalian cells have led to the creation of various hyperactive transposases such as SB11 and the latest one, SB100X, which shows a 100-fold higher activity compared to the original SB10.⁵ 

Using hyperactive (SB11) and “superactive” (SB100X) transposases, Xue and colleagues tested for stable gene transfer and expression in CB-derived CD34⁺ HSPCs.⁶  They report that SB100X is superior to SB11 (and the PB system) in transposing nakedly transfected transgenes (from a plasmid) to the host genome. Tested by colony formation assays (lasting for 14 days), long-term cultures for T, B, and natural killer lymphoid cells (lasting for weeks), and in vivo engraftment and differentiation assays (3 months in immunodeficient mice), DNA transposition and stable transgene expression were readily detected when SB100X was included in a transfection. Transgene expression was detected in both lymphoid and myeloid progeny from transfected and transplanted CD34⁺ HSPCs, although percentages of engrafted human CD45⁺ cells expressing the reporter gene (5%-8%) were lower than those previously achieved using gammaretroviral or lentiviral vectors by us and others.^7,8  Independently of this work, the group that created SB100X also achieved stable gene integration in engrafting HSPCs assayed for up to 4 months after transplantation.⁵  Common integration (transposition) sites were found in lymphoid and myeloid progeny of SB transfected CD34⁺ HSPCs.⁵  They also confirmed that SB transposition in HSPCs occurs at both intergenic and intragenic (often in introns) regions at TA dinucleotide sites as observed in other studies. The position of SB transposon insertion is much less biased toward intragenic or promoter regions as seen with retroviral vectors. Together, these 2 studies provided strong evidence of plasmid-mediated stable gene transfer into multipotent human HSPCs via DNA transposition at more favorable chromosomal positions. Furthermore, it is unnecessary to activate the cell prior to DNA delivery, although the current method of transfection kills about half of the cells.^5,6 

Further improvements of gene transfer and expression are anticipated for SB and other transposon systems such as PB. PB may offer advantages such as a larger cargo load (> 14 kb, allowing the inclusion of more genes or larger regulatory elements), different insertion preferences, and the exclusive use of TTAA target sequence.^2-4  For some applications, the PB transposition offers an additional advantage: the integrated transposon can be precisely excised by re-expression of PB transposase transiently as shown by several groups in diverse systems. Although the current PB system is inferior for HSPCs to the SB system using the superactive SB100X, I predict that the PB transposase will be improved significantly soon. Together, these transposon systems offer hopes to achieve virus-free, long-term gene transfer and expression in HSPCs and other cell types for various forms of gene therapy.