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Gene delivery into hCD34+ hematopoietic stem/progenitor cells (HSPCs) using human immunodeficiency virus, type 1-derived lentiviral vectors (LVs) has several promising therapeutic applications. Numerous clinical trials are currently underway. However, the efficiency, safety, and cost of LV gene therapy could be ameliorated by enhancing target cell transduction levels and reducing the amount of LV used on the cells. Several transduction enhancers already exist, such as fibronectin fragments or cationic compounds. Recently, we discovered Vectofusin-1, a new transduction enhancer, also called LAH4-A4, a short histidine-rich amphipathic peptide derived from the LAH4 family of DNA transfection agents. Vectofusin-1 enhances the infectivity of lentiviral and γ-retroviral vectors pseudotyped with various envelope glycoproteins. In this study, we compared a family of Vectofusin-1 isomers and showed that Vectofusin-1 remains the lead peptide for HSPC transduction enhancement with LVs pseudotyped with vesicular stomatitis virus glycoproteins and also with modified gibbon ape leukemia virus glycoproteins. By comparing the capacity of numerous Vectofusin-1 variants to promote the modified gibbon ape leukemia virus glycoprotein-pseudotyped lentiviral vector infectivity of HSPCs, the lysine residues on the N-terminal extremity of Vectofusin-1, a hydrophilic angle of 140° formed by the histidine residues in the Schiffer-Edmundson helical wheel representation, hydrophobic residues consisting of leucine were all found to be essential and helped to define a minimal active sequence. The data also show that the critical determinants necessary for lentiviral transduction enhancement are partially different from those necessary for efficient antibiotic or DNA transfection activity of LAH4 derivatives. In conclusion, these results help to decipher the action mechanism of Vectofusin-1 in the context of hCD34+ cell-based gene therapy. [ABSTRACT FROM AUTHOR]

[Display omitted] The use of cationic polymers in gene therapy aims at improving the circulation, protection and delivery of nucleic acids. The modification of poly(L-lysine) (PLL), including the insertion of hydrophobic moieties, is an efficient strategy to enhance the transfection efficiency of this synthetic delivery system. PLL is usually modified through either the extremities of the polymer chains or the amino groups present on the side chains. Herein the synthesis of dodecalysines as model of PLL with hexyl spacing units regularly distributed along the oligomer backbone is reported. Oligolysines terminated with azidohexyl groups were prepared by solid-phase synthesis and ligated by copper-assisted alkyne-azide cycloaddition using propargylamine. The iterative ligation of α-azidohexyloligolysines was more efficient when performed on resin as compared to the ligation in solution. The plasmid DNA transfection efficiency of the hydrophobically modified dodecalysines was then compared to that of unmodified dodecalysine. The results show that when formulated with the helper lipid 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine (DOPE) the hydrophobized dodecalysines exhibited a higher efficiency than the unmodified one. Interestingly, the efficiency could also be increased by the addition of a fusogenic peptide, which paves the way for modifications of the oligolysines that could lead to improved activities. [ABSTRACT FROM AUTHOR]

Small interfering RNAs (siRNAs) can down-regulate the expression of a target mRNA molecule in a sequence-specific manner, making them an attractive new class of drugs with broad potential for the treatment of diverse human diseases. Here, we report the synthesis of a series of cationic amphiphiles which were obtained by the coupling of amino acids and dipeptides onto a lipidic double chain. The new amphiphiles presenting a peptidic motif on a short hydrophilic spacer group were evaluated for selective gene silencing through RNA interference. Our results show that tryptophan residues boost siRNA delivery in an unexpected manner. The silencing experiments performed with very low concentrations of siRNA showed that the best formulations could induce significant death of tumor cells after silencing of polo-like kinase 1 which is implicated in cell cycle progression. In addition, these Trp containing peptide amphiphiles were highly efficient siRNA delivery vectors even in presence of competing serum proteins. [ABSTRACT FROM AUTHOR]

Gene delivery to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. In addition, muscle is an attractive target tissue because it is easily accessible. However, very few synthetic vectors proved capable of surpassing naked DNA mediated muscle gene transfer. In fact, only neutral copolymers, in particular poloxamers, demonstrated capacities to increase transgene expression in skeletal muscles. Here, we studied in vitro and in vivo behaviour of different bile salts. We report that sodium deoxycholate (DOC) and derivatives thereof increase after intramuscular injection by more than 100-fold the levels of the reporter gene luciferase compared to naked DNA. Using a LacZ expression cassette, we found that more than 20% of the muscle fibers expressed the reporter gene. Prolonged expression of a secreted reporter gene derived from a natural murine alkaline phosphatase enzyme could be documented. Altogether, our results demonstrate that bile salts belong to the most efficient chemicals identified so far for skeletal muscle gene transfer. Importantly, since these compounds are naturally found in the body, there is no risk of immune response against them and in addition several bile salts are already used in human medicine. Bile salt mediated muscle gene transfer may thus have broad applications in gene therapy. [ABSTRACT FROM AUTHOR]

Cationic carbon dots were fabricated by pyrolysis of citric acid and bPEI25k under microwave radiation. Various nanoparticles were produced in a 20–30% yield through straightforward modifications of the reaction parameters (stoichiometry of the reactants and energy supply regime). Particular attention was paid to the purification of the reaction products to ensure satisfactory elimination of the residual starting polyamine. Intrinsic properties of the particles (size, surface charge, photoluminescence and quantum yield) were measured and their ability to form stable complexes with nucleic acid was determined. Their potential to deliver plasmid DNA or small interfering RNA to various cell lines was investigated and compared to that of bPEI25k. The pDNA in vitro transfection efficiency of these carbon dots was similar to that of the parent PEI, as was their cytotoxicity. The higher cytotoxicity of bPEI25k/siRNA complexes when compared to that of the CD/siRNA complexes however had marked consequences on the gene silencing efficiency of the two carriers. These results are not fully consistent with those in some earlier reports on similar nanoparticles, revealing that toxicity of the carbon dots strongly depends on their protocol of fabrication. Finally, these carriers were evaluated for in vivo gene delivery through the non-invasive pulmonary route in mice. High transgene expression was obtained in the lung that was similar to that obtained with the golden standard formulation GL67A, but was associated with significantly lower toxicity. Post-functionalization of these carbon dots with PEG or targeting moieties should significantly broaden their scope and practical implications in improving their in vivo transfection efficiency and biocompatibility. [ABSTRACT FROM AUTHOR]

The mRNA vaccine technology has promising applications to fight infectious diseases as demonstrated by the licensing of two mRNA-based vaccines, Comirnaty® (Pfizer/BioNtech) and Spikevax® (Moderna), in the context of the Covid-19 crisis. Safe and effective delivery systems are essential to the performance of these vaccines and lipid nanoparticles (LNPs) able to entrap, protect and deliver the mRNA in vivo are considered by many as the current "best in class". Nevertheless, current mRNA/LNP vaccine technology has still some limitations, one of them being thermostability, as evidenced by the ultracold distribution chain required for the licensed vaccines. We found that the thermostability of mRNA/LNP, could be improved by a novel imidazole modified lipid, DOG-IM4, in combination with standard helper lipids. DOG-IM4 comprises an ionizable head group consisting of imidazole, a dioleoyl lipid tail and a short flexible polyoxyethylene spacer between the head and tail. Here we describe the synthesis of DOG-IM4 and show that DOG-IM4 LNPs confer strong immunization properties to influenza HA mRNA in mice and macaques and a remarkable stability to the encapsulated mRNA when stored liquid in phosphate buffered saline at 4 °C. We speculate the increased stability to result from some specific attributes of the lipid's imidazole head group. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract: In this study, the ability of synthetic fluorinated lipospermines to bind DNA and siRNA was investigated and the transfection efficiency and toxicity of the resulting lipoplexes in cell lines were evaluated. Three lipopolyamines displaying fluorous tags close to their cationic polar head (“HFP” polyamines) were synthesized. Their ability to condense pDNA and siRNA, and to form nanoparticles were characterized. Lipoplex stability was investigated in the presence of different surface active compounds and was shown to be significantly improved due to the presence of the fluorous tags. Transfection efficiencies were studied in HepG2 and 911 cell lines, and compared to that of DOGS, DOTAP, and Lipofectamine™ 2000. Also, the ability of these compounds to deliver nucleic acids into cells in the presence of high concentration of serum was quantified. By incorporating two fluorous tags in the direct vicinity of the polycationic head group of the lipospermines, we show efficient pDNA and siRNA formulation, and delivery to cultured cells. Fluorinated lipoplexes exhibit improved stability in the presence of amphiphilic compounds and retain high transfection efficiency in the presence of 50–75% serum. These results demonstrate that lipospermines displaying fluorous tags close to their cationic polar head bind to and deliver pDNA and siRNA with high cell viability in different cell lines. They are efficient non-viral vectors that exhibit remarkable serum compatibility. [Copyright &y& Elsevier]

Designed histidine-rich amphipathic cationic peptides, such as LAH4, have enhanced membrane disruption and antibiotic properties when the peptide adopts an alignment parallel to the membrane surface. Although this was previously achieved by lowering the pH, here we have designed a new generation of histidine-rich peptides that adopt a surface alignment at neutral pH. In vitro, this new generation of peptides are powerful antibiotics in terms of the concentrations required for antibiotic activity; the spectrum of target bacteria,fungi,and parasites;and the speed with which they kill. Further modifications to the peptides, including the addition of more hydrophobic residues at the N terminus, the inclusion of a helix-breaking proline residue or using D-amino acids as building blocks, modulated the biophysical properties of the peptides and led to substantial changes in toxicity to human and parasite cells but had only a minimal effect on the antibacterial and antifungal activity. Using a range of biophysical methods, in particular solid-state NMR, we show that the peptides are highly efficient at disrupting the anionic lipid component of model membranes. However, we also show that effective pore formation in such model membranes may be related to, but is not essential for, high antimicrobial activity by cationic amphipathic helical peptides. The information in this study comprises a new layer of detail in the understanding of the action of cationic helical antimicrobial peptides and shows that rational design is capable of producing potentially therapeutic membrane active peptides with properties tailored to their function. [ABSTRACT FROM AUTHOR]

Discusses a study which sought to determine whether the karyophilic properties of viral protein R (VPR), as well as the ability to bind nucleic acids, could be used to deliver various cell lines. Background on the C-terminal domain of Vpr which is able to transfect various cell lines; Shortest active sequence for gene tranfer; DNA binding studies and permeabilization assays performed in the study.