PDNAS

NaOH solution is added dropwise to an aqueous suspension of this ester at 40—70°C over 1 h and the reaction mixture kept for 2 h to give 86.6% DHNA of 98.7% purity (74), which is then esterified with (CgH O) to obtain PDNA. The esterification process is dramatically improved by adding a small amount of inorganic or organic acid, preferably methanesulfonic acid, benzene sulfonic acid, or naphthalene sulfonic acid subsequent isolation and crystallisation gives a pure product (75). 

Propylene Dinitramine (PDNA). See under Diaminopropane and Derivatives in Vol 5, D1143-R... 

Comment It is critical to remove excess cap analogue from in vitro transcribed transcripts prior to transfection, because the cap analogue will compete with transcripts for the cellular translational machinery. Also, even after DNase treatment, the RNA sample may still contain traces of functional pDNA, which may interfere with subsequent detection by RT-PCR. Furthermore, plasmids containing a mammalian promoter may even give rise to de novo transcription in transfected cells. 

Variations The poly(A) tailing kit (Ambion) produces a mRNA population with varying lengths of poly(A) tails, controlled by altering poly(A) polymerase concentrations and incubation times. An alternate method to incorporate a poly(A) tail is to clone a defined stretch of adenosines/ thymidines into the > UTR of the template pDNA. To allow transcripts to finish on an adenosine, the insert should be followed by a restriction site for an enzyme that cleaves 5 of the last antisense strand thymidine, such as Nsi I. In this way, the poly (A) tail can be incorporated directly into the... 

As pDNA and mRNA transfection differ in both the timing of mRNA expression and the gross amount of mRNA delivered to the cell, it is important to identify a suitable time point to measure miR-mediated repression. We observe that at any time point after transfection, pDNA transfections have higher measurable levels of miR-mediated repression compared to mRNA transfections (Fig. 6.2C). This difference may, in part, reflect a time lag of active miR-protein-complex formation relative to the onset of translation of the transfected Renilla luciferase mRNA. For single time point experiments, we decided to measure miR-mediated repression in mRNA and DNA transfections at 16 and 24 h, respectively. 

Figure 6.2 Critical parameters of the miR/mRNA co-transfection method. (A) Titration of mRNA amount. HeLa cells were transfected with increasing amounts of cap tail R-luc-4 sites mRNA and a fixed amount of firefly (F-luc) mRNA. R-luc expression (luciferase activity) was measured 5 h after transfection. (B) Titration of miCXCR4 concentration. HeLa cells were transfected with cap tail R-luc-4 sites mRNA, F-luc mRNA, and varying concentrations of miCXCR4. Luciferase activity was measured 16 h after transfection and fold-repression by the miR was calculated as in Fig. 6.1D (C) Time-course of miR-mediated repression. HeLa cells were co-transfected with cap tail R-luc-4 sites and F-luc (control) mRNAs, either with or without miCXCR4, and harvested at different time points. Repression was calculated as detailed in Fig. 6.1 and plotted against time (mRNA transfection data series depicted by the circles). Analogous plasmid DNA transfections are shown for reference (pDNA, diamonds). Averaged results from several experiments are shown with standard deviation. Data were previously published (Humphreys etal., 2005). Copyright PNAS, reprinted with permission.

An example of miR-dependent deadenylation of mRNA measured by this method is shown in Fig. 6.3C. In this case, HeLa cells (which express let-7) were transfected with a pDNA R-luc construct encoding three let-7 binding sites in the 3 UTR (3 X bulge), or with control constructs encoding either no sites (plasmid) or three mutated let-7 sites (3 x bulge mut) (constructs described in Pillai et al, 2005). Cells were harvested 24 h after transfection, RNA was purified for the PAT assay, and luciferase activity was measured from cell lysates. As reported previously, the presence of functional let-7 target sites results in specific repression of luciferase expression with very minor effects on mRNA stability (Pillai et al., 2005). The experiment in Fig. 6.3C demonstrates that the let-7 targeted reporter mRNA is selectively deadenylated. 

Polyplex surface shielding solves several crucial problems, but may also create new problems. Shielding can strongly reduce the efficiency of subsequent cellular steps of the delivery process [68, 69], and also can negatively alter other polyplex characteristics. For pDNA/PEI polyplexes with optimum medium size of PEI, PEG was found to reduce the polyplex stability in vivo [64, 65, 81]. For a discussion of these aspects see Sect. 3.1. 

The neoangiogenic tumor vasculature overexpresses certain integrins and other surface markers, which can also be used for targeting of polyplexes. The RGD peptide motif has been successfully applied for integrin-targeted pDNA [125-128] and siRNA [129, 130] delivery. In many cases, the PEG motif-containing peptide was attached to the polycation via a PEG spacer. For RGD-PEG-PEI/pDNA polyplexes, an optimum grafting with RGD-PEG was required because transfection... 

Intracellular pathways after escape from the endolysosomal system into the cytosol are less clear. Obvious bottlenecks include, in the case of gene transfer (pDNA delivery), cytosolic transport to the perinuclear area, nuclear uptake, and nuclear presentation of the pDNA to the transcriptional machinery in bioactive form. In the case of siRNA (or mRNA and some other nucleic acids such as oligonucleotides), cytosolic accessibility for the required function is essential. Besides cytosolic transport [176, 177] and the nuclear import of large nucleic acid molecules [178-180], incorporation of functional nuclear import peptide domains has been evaluated [181-186]. Another bottleneck, nucleic acid unpackaging [187], i.e., partial or complete dissociation from the polymeric carrier, which is required for biological accessibility of the delivered nucleic acid, will be discussed in Sect. 3.3. 

Fig. 1 Bioresponsive polyplexes. (a) Systemic circulation of shielded polyplexes in blood stream and attachment to cell surface receptor (b) endocytosis into endosomes, deshielding by cleavage of PEG linkers and activation of membrane-destabilizing component by acidic pH or other means (c) endosomal escape into cytosol (d) siRNA transfer to form a cytosolic RNA-induced silencing complex complex (e) cytosolic migration and intranuclear import of pDNA (/) presentation of pDNA in accessible form to the transcription machinery...

A different pH-triggered deshielding concept with hydrophilic polymers is based on reversing noncovalent electrostatic bonds [78, 195, 197]. For example, a pH-responsive sulfonamide/PEl system was developed for tumor-specific pDNA delivery [195]. At pH 7.4, the pH-sensitive diblock copolymer, poly(methacryloyl sulfadimethoxine) (PSD)-hZocA -PEG (PSD-b-PEG), binds to DNA/PEI polyplexes and shields against cell interaction. At pH 6.6 (such as in a hypoxic extracellular tumor environment or in endosomes), PSD-b-PEG becomes uncharged due to sulfonamide protonation and detaches from the nanoparticles, permitting PEI to interact with cells. In this fashion PSD-b-PEG is able to discern the small difference in pH between normal and tumor tissues. 

Tumor tissues overexpress matrix metalloproteinases (MMPs). A liposomal pDNA carrier (MEND) was developed containing PEG conjugated to lipid via a peptide linker that is a target sequence for MMPs. In this strategy, PEG is removed from the carrier via MMP-triggered cleavage [198]. Intravenous administration in... 

High nucleic acid/polymer affinity does not necessarily directly correlate with high efficiency. Apparently, an optimum has to be reached. Also, the big difference in size of different medical nucleic acids (pDNA has several thousand negative... 

On the other hand, pDNA/PEI polyplexes were found to be not stable enough in the extracellular in vivo environment. Unpackaging of PEI and PEG-PEI polyplexes was observed [64, 65, 81], for example by serum proteins, soluble glycosaminoglycans, or extracellular matrix components. The situation is even worse in the case of siRNA polyplexes, where PEI polyplexes are dissociated in full human serum, as monitored by fluorescence fluctuation spectroscopy [66, 67]. 

First clinical human gene therapy trials with polyplexes were performed using cancer vaccines based on autologous patient tumor cells. These were modified ex vivo with interleukin-2 pDNA. To obtain high level transfection rates of patient s primary tumor cells, Tf-PLL/pDNA polyplexes linked with inactivated endosomolytic adenovirus particles were applied [221]. Polymer-based in vivo human gene transfer studies were performed with PEGylated PLL polyplexes, delivering CFTR pDNA to the airway epithelium of cystic fibrosis patients [222],... 

Intravesical infusion of linear PEI/pDNA polyplexes was evaluated in patients with superficial bladder cancer where intravesical therapy with bacillus Calmette-Guerin had failed [6, 224]. Patients had low grade superficial bladder cancer, which expressed H19. The therapeutic pDNA contains H19 gene regulatory sequences that drive the expression of an intracellular toxin. Escalating doses of 2-20 mg plasmid per intravesical treatment were applied, with responders continuing to receive polyplexes once a month every month for 1 year. The treatment resulted in complete ablation of the marker tumor, without any new tumors in four of the 18 patients (22% overall complete response rate). Eight of the 18 patients (44%) had complete marker tumor ablation or a 50% reduction of the marker lesion. 

Systemic targeting of pDNA and siRNA polyplexes has been demonstrated in several animal models. In continuation of the work with localized antiproliferative and immunostimulatory poly(I C) RNA, intravenous systemic delivery of EGER-targeted PEG-modified polyplexes were successfully used for human carcinoma treatment in mice [225]. The therapeutic effect was most pronounced when intravenous delivery of poly(I C) polyplexes was followed by intraperitoneal injection of peripheral blood mononuclear cells [226]. This induced the complete cure of SCID mice with pre-established disseminated EGFR-overexpressing tumors, without adverse toxic effects. Due to the chemokines produced by the internalized poly (I C) in the tumor cells, the immune cells home to the tumors of the treated animal and contribute to the tumor destruction. 

EGER targeting was also used for systemic delivery of pDNA expressing the sodium iodide symporter (NIS) gene to liver cancer cells, followed by administration of radioactive isotope iodine-131, which accumulates in the tumor by NIS-mediated uptake in radiotherapeutic doses [227]. 

Various researchers have applied the receptor-targeted strategy in pharmacological models for tumor-targeted delivery of pDNA expressing tumor necrosis factor alpha (TNFa). For example, Tf- or Tf-PEG-shielded PEI polyplexes have been used... 

Prevette LE, Kodger TE, Reineke TM, Lynch ML (2007) Deciphering the role of hydrogen bonding in enhancing pDNA-polycation interactions. Langmuir 23 9773-9784... 

Kurosaki T, Kitahara T, Fumoto S, Nishida K, Nakamura J, Niidome T, Kodama Y, Nakagawa H, To H, Sasaki H (2009) Ternary complexes of pDNA, polyethylenimine, and gamma-polyglutamic acid for gene delivery systems. Biomaterials 30 2846-2853... 

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