Genetic recombination of poly(l-lysine) functionalized apoferritin nanocages that resemble viral capsid nanometer-sized platforms for gene therapy.

Currently, bioengineered apoferritin nanocages with flexible protein shells and functionalized modifications have become an attractive approach for efficient anti-tumor therapy. Here, we modified the N-terminus of H-chain subunits in apoferritin with different amounts of lysine via genetic recombination to obtain a poly(l-lysine) modified H-chain apoferritin (nL-HFn) nanocage for siRNA delivery and gene therapy. To achieve excellent cellular affinity and uptake, the nanocarriers were internalized through transferrin receptor-mediated endocytosis, then escaped from the endosome for cytoplasmic transport. Compared with natural apoferritin, the siRNA-loaded genetic recombination NPs modified with lysine exhibit stronger RNA-interference and antitumor efficiency both in vitro and in 4T1 tumor model mice. Therefore, bioengineered apoferritin nanocages modified with lysine might be a promising platform for nucleic acid drug delivery.

A novel prenyl-polybasic domain code determines lipid-binding specificity of the K-Ras membrane anchor.

Ras proteins must localize to the plasma membrane (PM) for biological function. The membrane anchor of the K-Ras4B isoform comprises a farnesylated and methylated C-terminal cysteine together with an adjacent hexa-lysine polybasic domain (PBD). Traditionally, polybasic sequences have been thought to interact electrostatically with negatively charged membranes showing no specificity for anionic lipid head groups. By contrast we recently showed that the K-Ras membrane anchor actually exhibits a very high degree of specificity for phosphatidylserine (PtdSer). The selectivity for PtdSer is determined by a combinatorial code comprising the PBD sequence plus the prenyl anchor. Lipid binding specificity is therefore altered by PBD point mutations that in turn modulate signaling output. For example, mutating Lys177 or Lys178 to glutamine switches K-Ras4B lipid affinity from PtdSer to phosphoinositol 4,5-bisphosphate (PIP2). Changing the lipid anchor from farnesyl to geranylgeranyl or the PBD lysines to arginines also changes lipid binding specificity. All-atom molecular dynamics simulations reveal the structural basis for these K-Ras anchor lipid-binding preferences. Here we examine the PM interactions of a series of geranylgeranylated PBD mutants and provide further evidence that the precise PBD sequence and prenyl lipid determines lipid sorting specificity of the K-Ras anchor and hence biological function.

A dual role of the ribosome-bound chaperones RAC/Ssb in maintaining the fidelity of translation termination.

The yeast ribosome-associated complex RAC and the Hsp70 homolog Ssb are anchored to the ribosome and together act as chaperones for the folding and co-translational assembly of nascent polypeptides. In addition, the RAC/Ssb system plays a crucial role in maintaining the fidelity of translation termination; however, the latter function is poorly understood. Here we show that the RAC/Ssb system promotes the fidelity of translation termination via two distinct mechanisms. First, via direct contacts with the ribosome and the nascent chain, RAC/Ssb facilitates the translation of stalling-prone poly-AAG/A sequences encoding for polylysine segments. Impairment of this function leads to enhanced ribosome stalling and to premature nascent polypeptide release at AAG/A codons. Second, RAC/Ssb is required for the assembly of fully functional ribosomes. When RAC/Ssb is absent, ribosome biogenesis is hampered such that core ribosomal particles are structurally altered at the decoding and peptidyl transferase centers. As a result, ribosomes assembled in the absence of RAC/Ssb bind to the aminoglycoside paromomycin with high affinity (KD = 76.6 nM) and display impaired discrimination between stop codons and sense codons. The combined data shed light on the multiple mechanisms by which the RAC/Ssb system promotes unimpeded biogenesis of newly synthesized polypeptides.

Deletion of eIF2ß lysine stretches creates a dominant negative that affects the translation and proliferation in human cell line: A tool for arresting the cell growth.

BACKGROUND: Eukaryote initiation factor 2 subunit ß (eIF2ß) plays a crucial role in regulation protein synthesis, which mediates the interaction of eIF2 with mRNA. eIF2ß contains evolutionarily conserved polylysine stretches in amino-terminal region and a zinc finger motif in the carboxy-terminus. METHODS: The gene eIF2ß was cloned under tetracycline transcription control and the polylysine stretches were deleted by site-directed mutagenesis (eIF2ßΔ3K). The plasmid was transfected into HEK 293 TetR cells. These cells were analyzed for their proliferative and translation capacities as well as cell death rate. Experiments were performed using gene reporter assays, western blotting, flow cytometry, cell sorting, cell proliferation assays and confocal immunofluorescence. RESULTS: eIF2ßΔ3K affected negatively the protein synthesis, cell proliferation and cell survival causing G2 cell cycle arrest and increased cell death, acting in a negative dominant manner against the native protein. Polylysine stretches are also essential for eIF2ß translocated from the cytoplasm to the nucleus, accumulating in the nucleolus and eIF2ßΔ3K did not make this translocation. DISCUSSION: eIF2ß is involved in the protein synthesis process and should act in nuclear processes as well. eIF2ßΔ3K reduces cell proliferation and causes cell death. Since translation control is essential for normal cell function and survival, the development of drugs or molecules that inhibit translation has become of great interest in the scenario of proliferative disorders. In conclusion, our results suggest the dominant negative eIF2ßΔ3K as a therapeutic strategy for the treatment of proliferative disorders and that eIF2ß polylysine stretch domains are promising targets for this.

Effects of Chromosomal Integration of the Vitreoscilla Hemoglobin Gene (vgb) and S-Adenosylmethionine Synthetase Gene (metK) on ε-Poly-L-Lysine Synthesis in Streptomyces albulus NK660.

ε-Poly-L-lysine (ε-PL) is a widely used natural food preservative. To test the effects of the Vitreoscilla hemoglobin (VHb) and S-adenosylmethionine (SAM) on ε-PL synthesis in Streptomyces albulus NK660, the heterologous VHb gene (vgb) and SAM synthetase gene (metK) were inserted into the S. albulus NK660 chromosome under the control of the constitutive ermE* promoter. CO-difference spectrum analysis showed S. albulus NK660-VHb strain could express functional VHb. S. albulus NK660-VHb produced 26.67 % higher ε-PL and 14.57 % higher biomass than the wild-type control, respectively. Reversed-phase high-pressure liquid chromatography (RP-HPLC) results showed the overexpression of the metK gene resulted in increased intracellular SAM synthesis in S. albulus NK660-SAM, which caused increases of biomass as well as the transcription level of ε-PL synthetase gene (pls). Results indicated that the expression of vgb and metK gene improved on ε-PL synthesis and biomass for S. albulus NK660, respectively.

Anti-angiogenic therapy increases intratumoral adenovirus distribution by inducing collagen degradation.

Conditionally replicating adenoviruses (CRAd) are a promising class of gene therapy agents that can overcome already known glioblastoma (GBM) resistance mechanisms but have limited distribution upon direct intratumoral (i.t.) injection. Collagen bundles in the extracellular matrix (ECM) have an important role in inhibiting virus distribution. In fact, ECM pre-treatment with collagenases improves virus distributions to tumor cells. Matrix metalloproteinases (MMPs) are an endogenous class of collagenases secreted by tumor cells whose function can be altered by different drugs including anti-angiogenic agents, such as bevacizumab. In this study we hypothesized that upregulation of MMP activity during anti-angiogenic therapy can improve CRAd-S-pk7 distribution in GBM. We find that MMP-2 activity in human U251 GBM xenografts increases (*P=0.03) and collagen IV content decreases (*P=0.01) during vascular endothelial growth factor (VEGF-A) antibody neutralization. After proving that collagen IV inhibits CRAd-S-pk7 distribution in U251 xenografts (Spearman rho=-0.38; **P=0.003), we show that VEGF-blocking antibody treatment followed by CRAd-S-pk7 i.t. injection reduces U251 tumor growth more than each individual agent alone (***P<0.0001). Our data propose a novel approach to improve virus distribution in tumors by relying on the early effects of anti-angiogenic therapy.

Ribosome-associated complex and Ssb are required for translational repression induced by polylysine segments within nascent chains.

When a polyadenylated nonstop transcript is fully translated, a complex consisting of the ribosome, the nonstop mRNA, and the C-terminally polylysine-tagged protein is generated. In Saccharomyces cerevisiae, a 3-step quality control system prevents formation of such dead-end complexes. Nonstop mRNA is rapidly degraded, translation of nonstop mRNA is repressed, and finally, nonstop proteins are cotranslationally degraded. Nonstop mRNA degradation depends on Ski7 and the exosome; nonstop protein degradation depends on the ribosome-bound E3 ligase Ltn1 and the proteasome. However, components which mediate translational repression of nonstop mRNA have previously not been identified. Here we show that the ribosome-bound chaperone system consisting of the ribosome-associated complex (RAC) and the Hsp70 homolog Ssb is required to stabilize translationally repressed ribosome-polylysine protein complexes, without affecting the folding or the degradation of polylysine proteins. As a consequence, in the absence of RAC/Ssb, polylysine proteins escaped translational repression and subsequently folded into their native conformation. This active role of RAC/Ssb in the quality control of polylysine proteins significantly contributed to the low level of expression of nonstop transcripts in vivo.

Effective gene delivery using stimulus-responsive catiomer designed with redox-sensitive disulfide and acid-labile imine linkers.

A dual stimulus-responsive mPEG-SS-PLL(15)-glutaraldehyde star (mPEG-SS-PLL(15)-star) catiomer is developed and biologically evaluated. The catiomer system combines redox-sensitive removal of an external PEG shell with acid-induced escape from the endosomal compartment. The design rationale for PEG shell removal is to augment intracellular uptake of mPEG-SS-PLL(15)-star/DNA complexes in the presence of tumor-relevant glutathione (GSH) concentration, while the acid-induced dissociation is to accelerate the release of genetic payload following successful internalization into targeted cells. Size alterations of complexes in the presence of 10 mM GSH suggest stimulus-induced shedding of external PEG layers under redox conditions that intracellularly present in the tumor microenvironment. Dynamic laser light scattering experiments under endosomal pH conditions show rapid destabilization of mPEG-SS-PLL(15)-star/DNA complexes that is followed by facilitating efficient release of encapsulated DNA, as demonstrated by agarose gel electrophoresis. Biological efficacy assessment using pEGFP-C1 plasmid DNA encoding green fluorescence protein and pGL-3 plasmid DNA encoding luciferase as reporter genes indicate comparable transfection efficiency of 293T cells of the catiomer with a conventional polyethyleneimine (bPEI-25k)-based gene delivery system. These experimental results show that mPEG-SS-PLL(15)-star represents a promising design for future nonviral gene delivery applications with high DNA binding ability, low cytotoxicity, and high transfection efficiency.

Use of Cre/loxP recombination to swap cell binding motifs on the adenoviral capsid protein IX.

We used Cre/loxP recombination to swap targeting ligands present on the adenoviral capsid protein IX (pIX). A loxP-flanked sequence encoding poly-lysine (pK-binds heparan sulfate proteoglycans) was engineered onto the 3'-terminus of pIX, and the resulting fusion protein allowed for routine virus propagation. Growth of this virus on Cre-expressing cells removed the pK coding sequence, generating virus that could only infect through alternative ligands, such as a tyrosine kinase receptor A (TrkA)-binding motif engineered into the capsid fibre protein for enhanced infection of neuronal cells. We used a similar approach to swap the pK motif on pIX for a sequence encoding a single-domain antibody directed towards CD66c for targeted infection of cancer cells; Cre-mediated removal of the pK-coding sequence simultaneously placed the single-domain antibody coding sequence in frame with pIX. Thus, we have developed a simple method to propagate virus lacking native viral tropism but containing cell-specific binding ligands.

Both the C-terminal polylysine region and the farnesylation of K-RasB are important for its specific interaction with calmodulin.

BACKGROUND: Ras protein, as one of intracellular signal switches, plays various roles in several cell activities such as differentiation and proliferation. There is considerable evidence showing that calmodulin (CaM) binds to K-RasB and dissociates K-RasB from membrane and that the inactivation of CaM is able to induce K-RasB activation. However, the mechanism for the interaction of CaM with K-RasB is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: Here, by applying fluorescence spectroscopy and isothermal titration calorimetry, we have obtained thermodynamic parameters for the interaction between these two proteins and identified the important elements of K-RasB for its interaction with Ca(2+)/CaM. One K-RasB molecule interacts with one CaM molecule in a GTP dependent manner with moderate, micromolar affinity at physiological pH and physiologic ionic strength. Mutation in the polybasic domain of K-Ras decreases the binding affinity. By using a chimera in which the C-terminal polylysine region of K-RasB has been replaced with that of H-Ras and vice versa, we find that at physiological pH, H-Ras-(KKKKKK) and Ca(2+)/CaM formed a 1:1 complex with an equilibrium association constant around 10(5) M(-1), whereas no binding reaction of K-RasB-(DESGPC) with Ca(2+)/CaM is detected. Furthermore, the interaction of K-RasB with Ca(2+)/CaM is found to be enhanced by the farnesylation of K-RasB. CONCLUSIONS/SIGNIFICANCE: We demonstrate that the polylysine region of K-RasB not only contributes importantly to the interaction of K-RasB with Ca(2+)/CaM, but also defines its isoform specific interaction with Ca(2+)/CaM. The farnesylation of K-RasB is also important for its specific interaction with Ca(2+)/CaM. Information obtained here can enhance our understanding of how CaM interacts with K-RasB in physiological environments.

Polycationic amino acid tags enhance soluble expression of Candida antarctica lipase B in recombinant Escherichia coli.

Lipase (EC 3.1.1.3) is a popular enzyme used as an ingredient in detergents and biocatalyst in many biochemical reactions. Lipase is usually expressed in Escherichia coli as an inactive inclusion body and at a low level. In this study, Candida antarctica lipase B (CalB) was fused with various polycationic amino acid tags and expressed in E. coli in order to increase a soluble expression level. By induction with 1.0 mM IPTG, the authentic and fused CalBs were expressed at 27-56% of total protein. The 10-arginine and 10-lysine tags fused at the C-terminal of CalB significantly increased the solubility of CalB by five- to ninefold, relative to the case of the authentic CalB expressed in a recombinant E. coli Origami 2™ (DE3) strain. Among a series of the C-terminal poly-arginine tags, the recombinant CalB combined with the 10-arginine tag (CalB-R10) possessed the highest lipase specific activity of 9.5 ± 0.03 U/mg protein, corresponding to a fourfold enhancement compared with the authentic CalB.

Polyglutamine expansion mutation yields a pathological epitope linked to nucleation of protein aggregate: determinant of Huntington's disease onset.

Polyglutamine (polyQ) expansion mutation causes conformational, neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. These diseases are characterized by the aggregation of misfolded proteins, such as amyloid fibrils, which are toxic to cells. Amyloid fibrils are formed by a nucleated growth polymerization reaction. Unexpectedly, the critical nucleus of polyQ aggregation was found to be a monomer, suggesting that the rate-limiting nucleation process of polyQ aggregation involves the folding of mutated protein monomers. The monoclonal antibody 1C2 selectively recognizes expanded pathogenic and aggregate-prone glutamine repeats in polyQ diseases, including Huntington's disease (HD), as well as binding to polyleucine. We have therefore assayed the in vitro and in vivo aggregation kinetics of these monomeric proteins. We found that the repeat-length-dependent differences in aggregation lag times of variable lengths of polyQ and polyleucine tracts were consistently related to the integration of the length-dependent intensity of anti-1C2 signal on soluble monomers of these proteins. Surprisingly, the correlation between the aggregation lag times of polyQ tracts and the intensity of anti-1C2 signal on soluble monomers of huntingtin precisely reflected the repeat-length dependent age-of-onset of HD patients. These data suggest that the alterations in protein surface structure due to polyQ expansion mutation in soluble monomers of the mutated proteins act as an amyloid-precursor epitope. This, in turn, leads to nucleation, a key process in protein aggregation, thereby determining HD onset. These findings provide new insight into the gain-of-function mechanisms of polyQ diseases, in which polyQ expansion leads to nucleation rather than having toxic effects on the cells.

Cardiomyocyte preparation, culture, and gene transfer.

Neonatal rat ventricular myocytes (NRVMs) cultured in vitro have been used as a model system for easily recreating and studying several cardiac molecular conditions, such as hypertrophy, oxygen deprivation, and gene expression. However, low efficiency of gene transfer has often represented one of the major limitations of this technique. In this chapter we describe in detail how to isolate NRVMs from neonatal rat heart and the optimal conditions for their long-term culture. Different cardiomyocyte transfection methodologies, based on viral or viral/chemical delivery carriers, are also discussed.

Identification of secretory granule phosphatidylinositol 4,5-bisphosphate-interacting proteins using an affinity pulldown strategy.

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) synthesis is required for calcium-dependent exocytosis in neurosecretory cells. We developed a PtdIns(4,5)P2 bead pulldown strategy combined with subcellular fractionation to identify endogenous chromaffin granule proteins that interact with PtdIns(4,5)P2. We identified two synaptotagmin isoforms, synaptotagmins 1 and 7; spectrin; alpha-adaptin; and synaptotagmin-like protein 4 (granuphilin) by mass spectrometry and Western blotting. The interaction between synaptotagmin 7 and PtdIns(4,5)P2 and its functional relevance was investigated. The 45-kDa isoform of synaptotagmin 7 was found to be highly expressed in adrenal chromaffin cells compared with PC12 cells and to mainly localize to secretory granules by subcellular fractionation, immunoisolation, and immunocytochemistry. We demonstrated that synaptotagmin 7 binds PtdIns(4,5)P2 via the C2B domain in the absence of calcium and via both the C2A and C2B domains in the presence of calcium. We mutated the polylysine stretch in synaptotagmin 7 C2B and demonstrated that this mutant domain lacks the calcium-independent PtdIns(4,5)P2 binding. Synaptotagmin 7 C2B domain inhibited catecholamine release from digitonin-permeabilized chromaffin cells, and this inhibition was abrogated with the C2B polylysine mutant. These data indicate that synaptotagmin 7 C2B-effector interactions, which occur via the polylysine stretch, including calcium-independent PtdIns(4,5)P2 binding, are important for chromaffin granule exocytosis.

Biological properties of photocrosslinked alginate microcapsules.

An alternative form of gene therapy using recombinant cell lines delivering therapeutic products encapsulated in alginate hydrogel has proven effective in treating many murine models. The lack of long-term capsule stability has led to a new strategy to reinforce the microcapsules with a photopolymerized interpenetrating covalent network of N-vinylpyrrolidone (NVP) and sodium acrylate. Here the properties for potential application in gene therapy are reported. In assessing potential toxicity of the unpolymerized residues, HPLC showed that even after 1 week of washing, no toxic monomers could be detected. Their ability to sustain cell growth was monitored with growth of the encapsulated cells in vitro and in vivo. Although the initial photopolymerization caused significant cell damage, the cells were able to recover normal growth rates thereafter. After implanting into mice, the NVP-modified capsules showed a high level of biocompatibility as measured by hematological and biochemical functional tests. There was also no difference in the amount and type of plasma proteins adsorbing to the NVP-modified and the classical alginate capsules, thus indicating their similar biological compatibility. Both in vitro and in vivo tests confirmed that the NVP-modified capsules were more resistant to osmotic stress than the alginate microcapsules. Furthermore, when applied to the treatment of a murine model of human cancer by delivering encapsulated cells secreting angiostatin, the NVP-modified microcapsules suppressed tumor growth as successfully as the regular alginate microcapsules. In conclusion, the covalently modified microcapsules have shown a high level of biocompatibility, safety, increase in stability, and clinical efficacy for use as immunoisolation devices in gene therapy.

Photochemical enhancement of gene delivery to glioblastoma cells is dependent on the vector applied.

BACKGROUND: Photodynamic therapy (PDT) and gene therapy protocols are separately under clinical evaluation for treatment of brain malignancies. Here, the potential of a novel combination technique, photo-induced delivery of macromolecules and genes to glioblastoma cells, is evaluated. MATERIALS AND METHODS: The photochemical effect on survival of GaMg and U-87Mg cells after incubation with the protein toxin gelonin, on transfection with a plasmid complexed to poly-L-lysine (PLL), and on transduction with adenovirus serotype 5 (Ad5) and adeno-associated virus type 5 (AAV5) vectors, were studied. RESULTS: Cytotoxicity of gelonin and gene transfer from plasmid/PLL complexes were considerably improved by photochemical treatment in both cell lines, while the light-inducible effect on Ad5 transduction was most pronounced in U-87Mg. For the first time, photochemical enhancement of AAV transduction is shown. A 4-fold increase in percentage positive cells was detected after photochemical treatment of AAV5-infected GaMg cells. However, in contrast to Ad5, AAV5 transduction of U-87Mg remained unaffected by light treatment, independently of viral dose, light dose and timing of the light treatment relative to the transduction period. CONCLUSION: Photochemical treatment is a versatile tool for macromolecular delivery to glioblastoma cells, however, the photochemical effect on gene transfer by viral vectors is highly dependent on the cell line and vector applied.

A new biodegradable poly-amino acid: alpha,beta-poly[(N-hydroxypropyl/aminoethyl)-DL-aspartamide-co-L-lysine], a potential nonviral vector for gene delivery.

A new class of biodegradable poly-amino acid, alpha,beta-poly[(N-hydroxypropyl/aminoethyl)-DL-Aspartamide-co-L-Lysine] (PHAAL), was synthesized by ring-opening of poly[succinimide-co-lysine](PSL) with n-propanolamine and ethylene diamine after thermal copolycondensation of DL-Aspartic acid and L-lysine under reduced pressure. Different ratio feeds of PSL were obtained and characterized by 1H-NMR, Fourier transformed infrared spectroscopy, X-ray, thermogravimetric analysis and gel permeation chromatography experiments. As one of the polycationic materials, performed for gene delivery carrier, the PHAAL degradation experiment was carried out in PBS (10 mM, pH =7.4) and enzyme (papain, trypsine 1 mg/ml, 37 +/- 0.1 degree C) solution. PHAAL had lower cytotoxicity than polyethylenimine (25KDa) and poly-L-Lysine (30 KDa), in Hela, E.C.V.-304, Bcap 37 cell lines. Particle size and zeta, potential of PHAAL/DNA complexes were measured. Sizes ranged from 300-500 nm and zeta potentials were at -20 to 2,5 mV. The condensation ability of PHAAL for DNA was evaluated by agarose gel electrophoresis. The PHAAL could completely neutralize DNA at N/P ratio (w/w) 150:1.

A multiprong approach to cancer gene therapy by coencapsulated cells.

Immune-isolation of nonautologous cells with microencapsulation protects these cells from graft rejection, thus allowing the same recombinant therapeutic cell line to be implanted in different recipients. This approach was successful in treating HER2/neu-expressing tumors in mice by delivering an interleukin-2 fusion protein (sFvIL-2), or angiostatin. However, treatment with interleukin-2 led to profuse inflammation, while angiostatin delivery did not result in long-term tumor suppression, in part due to endothelial cell-independent neovascularization (vascular mimicry). We hypothesize that coencapsulating the two producer cells in the same microcapsules may enhance the efficacy and ameliorate the above side effects. Hence, B16-F0/neu tumor-bearing mice were implanted with sFvIL-2- and angiostatin-secreting cells coencapsulated in the same alginate-poly-L-lysine-alginate microcapsules. However, this protocol only produced an incremental but not synergistic improvement, as measured with greater tumor suppression and improved survival. Compared to the single sFvIL-2 treatment, the coencapsulation protocol showed improved efficacy associated with: mobilization of sFvIL-2 from the spleen; a higher level of cytokine delivery systemically and to the tumors; increased tumor and tumor-associated endothelial cell apoptosis; and a reduced host inflammatory response. However, compared to the single angiostatin treatment, the efficacy was reduced, primarily due to a "bystander" effect in which the angiostatin-secreting cells suffered similar transgene silencing as the coencapsulated cytokine-secreting cells. Nevertheless, the level of "vascular mimicry" of the single angiostatin treatment was significantly reduced. Hence, while there was no synergy in efficacy, an incremental improvement and some reduction in undesirable side effects of inflammation and vascular mimicry were achieved over the single treatments.

Copolymers of poly-L-lysine with serine and tryptophan form stable DNA vectors: implications for receptor-mediated gene transfer.

Inefficient gene transfer and poor stability in physiological medium are important shortcomings for receptor-mediated gene transfer vectors. Here, we evaluate vectors formulated with random copolymers of L-lysine/L-serine (3:1) and L-lysine/L-tryptophan (4:1), focusing on both their biophysical and functional characterization. By means of dynamic light scattering (DLS) and transmission electron microscopy (TEM), we demonstrate that poly-L-lysine (pK), poly-L-lysine-L-tryptophan (pKW) and poly-L-lysine-L-serine (pKS) are able to form compacted, small particles when mixed with plasmid DNA in the absence of salt. Upon dilution in physiological medium, copolymers of both lys/ser and lys/trp do not aggregate, in contrast with poly-L-lysine DNA complexes as determined by scattering, DLS and TEM measurements. Tight packing, as demonstrated by resistance to heparin, SDS and trypsin treatments, is also featured in tryptophan-containing complexes. Successful receptor-mediated endocytosis gene transfer using galactosylated copolymers into cells expressing the asiagloglycoprotein receptor correlated with lack of aggregation. Particles obtained using galactosylated poly-L-lysine-L-tryptophan (Gal-pKW) copolymer demonstrated specific receptor-mediated gene transfer since reporter gene activity dropped in the presence of an excess ligand in the culture medium during transfection. Although copolymers of galactosylated poly-L-lysine-L-serine (Gal-pKS) do not aggregate in the presence of salt, they are not able to internalize in a specific receptor-mediated endocytosis fashion. The introduction of bulky aromatic/hydrophobic (tryptophan) or hydrophillic (serine) moieties into the positively charged vectors allows the compacted particles to disperse into salt-containing medium avoiding salt-induced aggregation. Moreover, tryptophan-containing particles are able to mediate specific gene transfer via receptor-mediated endocytosis.

CTb targeted non-viral cDNA delivery enhances transgene expression in neurons.

BACKGROUND: Efficient neuronal gene therapy is a goal for the long-term repair and regeneration of the injured central nervous system (CNS). We investigated whether targeting cDNA to neurons with cholera toxin b chain conjugated non-viral polyplexes led to increased efficiency of non-viral gene transfer in the CNS. Here, we illustrate the potential for this strategy by demonstrating enhanced transfection of a differentiated neuronal cell type, PC12. METHODS: In vitro transfection efficiency of a cholera toxin b chain-poly(D-lysine) molecular conjugate (CTb-K(100)) was compared by fluorescence-activated cell sorting (FACS) analysis of green fluorescent protein (GFP) expression and luminometric measurement of beta-galactosidase (beta-gal) expression, to untargeted poly(D-lysine) (K(100)) in undifferentiated and NGF-differentiated PC12 cells. RESULTS: Transfection of undifferentiated PC12 cells with CTb-K(100) polyplexes resulted in a 36-fold increase in levels of pCMV-DNA(LacZ) expression and a 20-fold increase in the frequency of transduction with pCMV-DNA(GFP), compared with untargeted K(100) polyplexes. Treatment of PC12 cells with 50 ng/ml/day of NGF for 14 days led to differentiation to a neuronal phenotype. Transfection of NGF-differentiated cells with CTb-K(100) polyplexes resulted in a 133-fold increase in levels of pCMV-DNA(LacZ) expression and a 11-fold increase in the percentage of cells transduced with pCMV-DNA(GFP), compared with untargeted K(100) polyplexes. Transfection was dependent on CTb, with CTb-K(100)-mediated transfections competitively inhibited with free CTb in both PC12 phenotypes. CONCLUSIONS: Non-viral systems for gene transfer in damaged CNS show superior toxicological profiles to most viruses but are limited by inefficient and non-selective gene expression in target tissue. Cholera toxin is known to interact preferentially with neuronal cells of the central and peripheral nervous systems, mediating binding through the b subunit, CTb, and the pentasaccharide moiety of the gangliosaccharide, GM1, which is present at high levels on the neuronal cell surface. Here, we show that a molecular conjugate of the CTb subunit, covalently linked to poly(D-lysine), is able to successfully target and significantly enhance transfection of a neuronal cell type, NGF-differentiated rat PC12 pheochromocytoma cells. This observation encourages the further development of non-viral strategies for the delivery of therapeutic genes to neurons.