Post-nuclear gene delivery events for transgene expression by biocleavable polyrotaxanes
Introduction
Transgene expression has long been proposed as an innovative therapeutic strategy for directly treating diseases at the level of genes, and considerable efforts have been made to improve the efficiency of protein production [1], [2], [3], [4], [5], [6], [7]. To successfully achieve this, an efficient and targeted gene delivery system is necessary. Non-viral vectors would be useful gene delivery systems, especially from the viewpoint of safety, although they have a very lower transfection activity compared to viral vectors. A previous comparison revealed a surprising difference in transfection activity between viral and non-viral vectors: this difference is largely due to the intranuclear disposition of DNA rather than its delivery to the nucleus [8], [9]. The concept of intranuclear disposition assumes the following processes: DNA release from a vector, transcription to mRNA and translation to proteins (Fig. 1A). This led us to the hypothesis that DNA release from a vector in the nucleus represents a critical and dominant event in transfection activity.
To date, we reported on the development of an innovative gene delivery system [10], [11], prepared by integrating a multifunctional envelope-type nano device (MEND) [5], [12], [13], [14] and a biocleavable polyrotaxane (DMAE-SS-PRX) [15], [16], [17] (Fig. 1B). The MEND consists of plasmid DNA (pDNA) particles condensed with a polycation, with the outer envelopes coated with a lipid, which mimics envelope-type viruses [5], [12], [13], [14]. An advanced MEND system modified with a cell-penetrating peptide, octaarginine (R8), (R8-MEND) showed a high transfection activity comparable to that of reported potent viral vectors [13], [14]. On the other hand, DMAE-SS-PRX is an artificial DNA condenser [15], [16], [17], which can condense pDNA and then release it under intracellular reductive conditions. We concluded that DMAE-SS-PRX would be an appropriate model polycation for investigating the relationship between the DNA release in nucleus and transfection activity, because the cationic density in a DMAE-SS-PRX-molecule can be easily controlled, thus permitting the efficiency of DNA release to be adjusted [16].
We previously demonstrated the existence of a close relationship between the efficiency of DNA release and the transfection activity, using an integrated system of R8-MEND and DMAE-SS-PRX [10], [11]. It is particularly noteworthy that, from the standpoint of transfection, our integration system, which is comprised of the R8-MEND with DMAE-SS-PRX, was 5-fold greater than that for a conventional R8-MEND with protamine, which is a natural DNA condenser that shows a high transfection activity [18], [19], [20]. In vitro DNA release experiments showed that pDNA was efficiently released from the condensed pDNA particles, when DMAE-SS-PRX with a high cationic density was used [11]. Moreover, we provide evidence to show that cationic density in DMAE-SS-PRX clearly has a positive influence on the efficiency of DNA release in the nucleus in living cells, resulting in high gene expression, as evidenced by imaging the nuclear condensation/decondensation status of pDNA using a new technology [10].
Based on the previous results, we hypothesized that a high cationic density in DMAE-SS-PRX would aid nuclear DNA release, resulting in strong transgene expression. In this study, we first screened the optimal DMAE-SS-PRX for nuclear DNA release. We next investigated the relationship between the efficiency of DNA release and transfection activity, and the results showed that the transfection activity was proportional to DNA release to some extent. Unexpectedly, transfection activity was decreased when the value for efficiency of DNA release was higher than a certain value. To clearly understand the intranuclear fate of condensed pDNA particles until gene expression could be achieved, we carried out a detailed investigation of the transgene process before and after the nuclear delivery of pDNA.
Section snippets
Materials
pDNA encoding enhanced green fluorescent protein and luciferase protein (pEGFPLuc) was obtained from BD Bioscience Clontech (Palo Alto, CA, USA). pDNA was purified using a Qiagen EndoFree Plasmid Mega Kit (Qiagen GmbH, Hilden, Germany). Phosphatidic acid (PA) was purchased from Sigma (St. Louis, MO, USA). 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was purchased from AVANTI Polar Lipids Inc. (Alabaster, AL, USA). Stearyl octaarginine (STR-R8) was obtained from KURABO INDUSTRIES LTD
Evaluation for efficiency of DNA release from condensed pDNA particles
The efficiency of DNA release from condensed pDNA particles was investigated using various types of DMAE-SS-PRXs with different numbers of cationic DMAE groups (Table 1). It is generally accepted that the number of amino groups in a cationic polymer are closely related to polyplex formation, polyplex stability against a counter polyanion, transfection activity and etc. [23], [24]. We previously confirmed that pDNA particles condensed with DMAE-SS-PRX efficiently released DNA and showed a high
Discussion
Maintaining the supercoiled form of pDNA when condensed pDNA nanoparticles are designed is a major issue in this field of study. Since it is known that free pDNA possesses some unique features; its supercoiled form is more efficient in gene expression than the linear and open circular forms, which can be damaged. Several studies showed that the supercoiled form of pDNA is susceptible to being converted into the linear or open circular form during microencapsulation, resulting in a significant
Conclusion
In this study, we demonstrated a close relationship between the efficiency of DNA release and transcription efficiency. We also found that when the efficiency of DNA release from condensed pDNA particles is too high, this constitutes a negative contribution to transfection activity. Based on these results and our conclusions, it was presumed that a large amount of free cations released from the condensed pDNA particles might inhibit the post-transcription process. This finding provides a new
Acknowledgements
This study was funded by Special Coordination Funds for Promoting Science and Technology of the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government (MEXT), and in part by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation, Japan (NIBIO), a Grant-in-Aid for Young Scientists (A) from MEXT. We also thank Dr. Milton Feather for his helpful advice in writing the manuscript.
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Cited by (0)
- 1
These authors contributed equally as first author.
- 2
Current address: Institute of Bioengineering and Nanotechnology, 31 Biopolis way, The Nanos, #07-01, 138-669, Singapore.