Neutral Block Copolymer Assisted Gene Delivery using Hydrodynamic Limb Vein Injection.

Three different amphiphilic block copolymer families are synthesized to investigate new opportunities to enhance gene delivery via Hydrodynamic Limb Vein (HLV) injections. First a polyoxazoline-based family containing mostly one poly(2-methyl-2-oxazoline) (PMeOx) block and a second block POx with an ethyl (EtOx), isopropyl (iPrOx) or phenyl substituent (PhOx) is synthesized. Then an ABC poly(2-ethyl-2-oxazoline)-b-poly(2-n-propyl-2-oxazoline)-b-poly(2-methyl-2-oxazoline) triblock copolymer is synthesized, with a thermosensitive middle block. Finally, polyglycidol-b-polybutylenoxide-b-polyglycidol copolymers with various molar masses and amphiphilic balance are produced. The simple architecture of neutral amphiphilic triblock copolymer is not sufficient to obtain enhanced in vivo gene transfection. Double or triple amphiphilic neutral block copolymers are improving the in vivo transfection performances through HLV administration as far as a block having an lower critical solution temperature is incorporated in the vector. The molar mass of the copolymer does not seem to affect the vector performances in a significant manner.

Impact of net charge, targeting ligand amount and mRNA modification on the uptake, intracellular routing and the transfection efficiency of mRNA lipopolyplexes in dendritic cells.

Targeting mRNA formulations to achieve cell specificity is one of the challenges that must be tackled to mettle their therapeutic potential. Here, lipopolyplexes (LPR) bearing tri-mannose-lipid (TM) are used to target mannose receptor on dendritic cells. We investigated the impact of the net charge and percentage of TM units on the binding, uptake, transfection efficiency (TE) and RNA sensors activation. Binding and uptake capacities of naked and targeted LPR increase with the percent of cationic lipid, but the latter are 2-fold more up taken by the cells. Cationic LPR bearing 5 % and 10 % TM were localized in acidic compartments in contrast to naked LPR and 2.5 % TM-LPR. The drawback is the dramatic decrease of TE as the number of TM-units increases. Cationic LPR bearing 5 % and 10 % TM strongly induced NF-κB and PKR phosphorylation at 6 h. Conversely, mTOR is less activated in line with their low TE. Those side effects are overcome by using 5-methoxyuridine mRNA resulting in an improved TE due to non-phosphorylation of NF-κB and PKR and mTOR activation. Our results point out that targeting DC via mannose receptor triggers a higher uptake of cationic LPRs and fast routing to acidic compartments, and that efficient TE requires low number of TM units use or modified mRNA to escape RNA sensors activation to enhance the translation.

CFTR and dystrophin encoding plasmids carrying both luciferase reporter gene, nuclear import specific sequences and triple helix sites.

Duchenne Muscular Dystrophy and Cystic Fibrosis are two major monogenetic diseases which could be treated by non-viral gene therapy. For this purpose, plasmid DNA (pDNA) coding for the functional genes requires its equipment with signal molecules favouring its intracellular trafficking and delivery in the nucleus of the target cells. Here, two novel constructions of large pDNAs encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and full-length dystrophin (DYS) genes are reported. The expression of CFTR and DYS genes are driven respectively by the hCEF1 airway epithelial cells and spc5-12 muscle cells specific promoter. Those pDNAs encode also the luciferase reporter gene driven by the CMV promoter to evaluate gene delivery in animals by bioluminescence. In addition, oligopurine • oligopyrimidine sequences are inserted to enable equipment of pDNAs with peptides conjugated with a triple helix forming oligonucleotide (TFO). Furthermore, specific κB sequences are also inserted to promote their NFκB-mediated nuclear import. pDNA constructions are reported; transfection efficiency, tissue specific expression of CFTR and dystrophin in target cells, and triple helix formation are demonstrated. These plasmids are tools of interest to develop non-viral gene therapy of Cystic Fibrosis and Duchenne Muscular Dystrophy.

Reversible stabilization of DNA/PEI complexes by reducible click-linkage between DNA and polymer. A new polyplex concept for lowering polymer quantity.

Nonviral transfection of mammalian cells can be performed with electrostatic complexes (polyplexes) between a plasmid DNA (pDNA) encoding a foreign gene and a cationic polymer. However, an excess of the cationic polymer is required for pDNA condensation and polyplexes formation, which generate in vivo toxicity. Here, we present a new concept of polyplexes preparation aiming to reduce the polymer quantity. pDNA was functionalized with 3,6,9-trioxaundecan-1- {4 - [(2-chloroethyl) ethylamino)] - benzylamino}, 11-azide, and polyethyleneimine (lPEI) with reducible dibenzocyclooctyl (SS-DBCO) groups allowing azide-alkyne cycloaddition between pDNA and lPEI after condensation. The size of polyplexes with DBCO-SS-lPEI was smaller than with lPEI due to a stronger DNA condensation thanks to linkages between polymer and pDNA preventing dissociation until disulfide bridges reduction. In vitro transfection showed that the amount of DBCO-SS-lPEI leading to the most efficient polyplexes was three times lower than lPEI. As expected, toxicity in mice was significantly reduced upon intravenous injection of DBCO-SS-lPEI polyplexes at doses where the lPEI polyplexes killed mice. This is probably due to the high stability of the DBCO-SS-lPEI polyplexes which prevented their aggregation in the pulmonary capillaries. Overall, this new concept of polyplexes with DBCO-SS-lPEI offering the possibility of administering higher doses of polyplexes than lPEI and their ability to pass the pulmonary barrier could be favorably exploited for transfection of distant organs or tissues, such as tumors.

Selective attachment of a microtubule interacting peptide to plasmid DNA via a triplex forming oligonucleotide for transfection improvement.

In nonviral gene therapy approaches, the linkage of signal molecules to plasmid DNA (pDNA) is of interest for guiding its delivery to the nucleus. Here, we report its linkage to a peptide (P79-98) mediating migration on microtubules by using a triplex-forming oligonucleotide (TFO). pDNA of 5 kbp and 21 kbp containing 6 and 36 oligopurine • oligopyrimidine sites (TH), respectively, inserted outside the luciferase gene sequence were used. TFO with a dibenzocyclooctyl (DBCO) group in 3' end comprising some Bridged Nucleic Acid bases was conjugated by click chemistry with the peptide carrying an azide function in the C-terminal end. We found the formation of 6 and 18 triplex with pDNA of 5 kbp and 21 kbp, respectively. A twofold increase of the transfection efficiency was observed in the hind-limbs upon Hydrodynamic Limb Vein (HLV) injection in mice of naked P79-98 -pDNA of 21 kbp. This work paves the way for the selective equipping of pDNA with intracellular targeting molecules while preserving the full expression of the encoded gene.

Synthesis of Double Hydrophilic Block Copolymers Poly(2-isopropyl-2-oxazoline-b-ethylenimine) and their DNA Transfection Efficiency.

Gene delivery is now a part of the therapeutic arsenal for vaccination and treatments of inherited or acquired diseases. Polymers represent an opportunity to develop new synthetic vectors for gene transfer, with a prerequisite of improved delivery and reduced toxicity compared to existing polymers. Here, the synthesis in a two-step's procedure of linear poly(ethylenimine-b-2-isopropyl-2-oxazoline) block copolymers with the linear polyethylenimine (lPEI) block of various molar masses is reported; the molar mass of the poly(2-isopropyl-2-oxazoline) (PiPrOx) block has been set to 7 kg mol-1 . Plasmid DNA condensation is successfully achieved, and in vitro transfection efficiency of the copolymers is at least comparable to that obtained with the lPEI of same molar mass. lPEI-b-PiPrOx block copolymers are however less cytotoxic than their linear counterparts. PiPrOx can be a good alternative to PEG which is often used in drug delivery systems. The grafting of histidine moieties on the lPEI block of lPEI-b-PiPrOx does not provide any real improvement of the transfection efficiency. A weak DNA condensation is observed, due to increased steric hindrance along the lPEI backbone. The low cytotoxicity of lPEI-b-PiPrOx makes this family a good candidate for future gene delivery developments.

DNA minicircles as novel STAT3 decoy oligodeoxynucleotides endowed with anticancer activity in triple-negative breast cancer.

Decoy technology is a versatile and specific DNA oligonucleotide-based targeting strategy of pathogenic transcription factors (TFs). Chemical modifications of linear decoy oligonucleotides have been made to decrease nuclease sensitivity because of the presence of free ends but at the cost of new limitations that affect their use as therapeutic drugs. Although a short DNA minicircle is a phosphodiester nucleic acid without free ends, its potential therapeutic activity as a TF decoy oligonucleotide has not yet been investigated. Here we describe the in vitro and in vivo activity of formulated 95-bp minicircles bearing one or several STAT3 binding sequences in triple-negative breast cancer (TNBC). Minicircles bearing one STAT3 binding site interacted specifically with the active form of STAT3 and inhibited proliferation, induced apoptosis, slowed down cell cycle progression, and decreased STAT3 target gene expression in human and murine TNBC cells. Intratumoral injection of STAT3 minicircles inhibited tumor growth and metastasis in a murine model of TNBC. Increasing the number of STAT3 binding sites resulted in improved anticancer activity, opening the way for a TF multitargeting strategy. Our data provide the first demonstration of minicircles acting as STAT3 decoys and show that they could be an effective therapeutic drug for TNBC treatment.

mRNA Lipoplexes with Cationic and Ionizable α-Amino-lipophosphonates: Membrane Fusion, Transfection, mRNA Translation and Conformation.

Cationic liposomes are attractive carriers for mRNA delivery. Here, mRNA lipoplexes (LX) were prepared with the cationic lipids α-aminolipophosphonate (3b) or imidazolium lipophosphoramidate (2) associated with various α-aminolipophosphonates co-lipids comprising protonable groups (imidazole or pyridine) and DOPE. Physicochemical parameters of liposomes and their membrane fusion activity were measured. LXs comprising either 3b- or 2- allowed transfection of ~25% and 40% of dendritic cells with low cytotoxicity, respectively; the efficiency increased up to 80% when 2 was combined with the imidazole-based co-lipid 1. The transfections were high with 3b/1, 3b/DOPE, 2/1 and 2/DOPE LXs. We observed that the transfection level was not well correlated with the acid-mediated membrane fusion activity of liposomes supposed to destabilize endosomes. The mRNA release from LXs and its translation capacity after release were studied for the most efficient LXs. The results showed that the more mRNA was condensed, the poorer the translation efficiency after release was. In contrast to DNA, circular dichroism performed on mRNA complexed with 2/DOPE revealed the presence of denatured mRNA in LXs explaining this lack of translation efficiency. This is an important parameter that should be stressed for the preparation of mRNA LXs with a conserved mRNA translation activity.

LentiRILES, a miRNA-ON sensor system for monitoring the functionality of miRNA in cancer biology and therapy.

A major unresolved challenge in miRNA biology is the capacity to monitor the spatiotemporal activity of miRNAs expressed in animal disease models. We recently reported that the miRNA-ON monitoring system called RILES (RNAi-inducible expression Luciferase system) implanted in lentivirus expression system (LentiRILES) offers unique opportunity to decipher the kinetics of miRNA activity in vitro, in relation with their intracellular trafficking in glioblastoma cells. In this study, we describe in detail the method for the production of LentiRILES stable cell lines and employed it in several applications in the field of miRNA biology and therapy. We show that LentiRILES is a robust, highly specific and sensitive miRNA sensor system that can be used in vitro as a single-cell miRNA monitoring method, cell-based screening platform for miRNA therapeutics and as a tool to analyse the structure-function relationship of the miRNA duplex. Furthermore, we report the kinetics of miRNA activity upon the intracranial delivery of miRNA mimics in an orthotopic animal model of glioblastoma. This information is exploited to evaluate the tumour suppressive function of miRNA-200c as locoregional therapeutic modality to treat glioblastoma. Our data provide evidence that LentiRILES is a robust system, well suited to resolve the activity of endogenous and exogenously expressed miRNAs from basic research to gene and cell therapy.

Intracellular Routing and Recognition of Lipid-Based mRNA Nanoparticles.

Messenger RNA (mRNA) is being extensively used in gene therapy and vaccination due to its safety over DNA, in the following ways: its lack of integration risk, cytoplasmic expression, and transient expression compatible with fine regulations. However, clinical applications of mRNA are limited by its fast degradation by nucleases, and the activation of detrimental immune responses. Advances in mRNA applications, with the recent approval of COVID-19 vaccines, were fueled by optimization of the mRNA sequence and the development of mRNA delivery systems. Although delivery systems and mRNA sequence optimization have been abundantly reviewed, understanding of the intracellular processing of mRNA is mandatory to improve its applications. We will focus on lipid nanoparticles (LNPs) as they are the most advanced nanocarriers for the delivery of mRNA. Here, we will review how mRNA therapeutic potency can be affected by its interactions with cellular proteins and intracellular distribution.

DNA nuclear targeting sequences for enhanced non-viral gene transfer: An in vitro and in vivo study.

An important bottleneck for non-viral gene transfer commonly relates to translocation of nucleic acids into the nuclear compartment of target cells. So-called 3NFs are optimized short nucleotide sequences able to interact with the transcription factor nuclear factor κB (NF-κB), which can enhance the nuclear import of plasmid DNA (pDNA) carrying such motifs. In this work, we first designed a consistent set of six pDNAs featuring a common backbone and only varying in their 3NF sequences. These constructions were then transfected under various experimental settings. In vitro, cationic polymer-assisted pDNA delivery in five human-derived cell lines showed the potential advantage of 3NF carrying pDNA in diverse cellular contexts. In vivo, naked pDNAs were hydrodynamically delivered to muscle hindlimbs in healthy mice; this direct accurate comparative (in the absence of any gene carrier) revealed modest but consistent trends in favor of the pDNAs equipped with 3NF. In summary, the results reported emphasize the implications of various parameters on NF-κB-mediated pDNA nuclear import; under specific conditions, 3NF can provide modest to substantial advantages for pDNA gene transfer, in vitro as well as in vivo. This study thus further underscores the potential of optimized nuclear import for more efficient non-viral gene transfer applications.

In Vivo bone tissue induction by freeze-dried collagen-nanohydroxyapatite matrix loaded with BMP2/NS1 mRNAs lipopolyplexes.

Messenger RNA (mRNA) activated matrices (RAMs) are interesting to orchestrate tissue and organ regeneration due to the in-situ and sustained production of functional proteins. However, the immunogenicity of in vitro transcribed mRNA and the paucity of proper in vivo mRNA delivery vector need to be overcome to exert the therapeutic potential of RAM. We developed a dual mRNAs system for in vitro osteogenesis by co-delivering NS1 mRNA with BMP2 mRNA to inhibit RNA sensors and enhance BMP-2 expression. Next, we evaluated a lipopolyplex (LPR) formulation platform for in vivo mRNA delivery and adapted the LPRs for RAM preparation. The LPR formulated BMP2/NS1 mRNAs were incorporated into an optimized collagen-nanohydroxyapatite scaffold and freeze-dried to prepare ready-to-use RAMs. The loaded BMP2/NS1 mRNAs lipopolyplexes maintained their spherical morphology in the RAM, thanks to the core-shell structure of LPR. The mRNAs release from RAMs lasted for 16 days resulting in an enhanced prolonged transgene expression period compared to direct cell transfection. Once subcutaneously implanted in mice, the BMP2/NS1 mRNAs LPRs containing RAMs (RAM-BMP2/NS1) induced significant new bone tissue than those without NS1 mRNA, eight weeks post implantation. Overall, our results demonstrate that the BMP2/NS1 dual mRNAs system is suitable for osteogenic engagement, and the freeze-dried RAM-BMP2/NS1 could be promising off-the-shelf products for clinical orthopedic practice.

CEBA: A new heterobifunctional reagent for plasmid DNA functionalization by click chemistry.

Here, we report the synthesis of 3,6,9-trioxaundecan-1-{4-[(2-Chloroethyl)Ethylamino)]-Benzylamino},11-Azide (CEBA). CEBA alkylates the N7 of guanine of DNA thanks its chloroethyl group and can be coupled by a strain-promoted azide-alkyne cycloaddition to an alkynylated molecule. The optimization of the alkylation level of pDNA reveals that the expression of the encoded gene is preserved when it is randomly modified with at most 1 CEBA molecule per 150 bp. We show that the azido group of CEBA allows the linkage via click chemistry of CEBA-pDNA with a fluorophore or a peptide containing a dibenzocyclooctyne (DBCO) function. This new heterobifunctional reagent opens new ways to equip pDNA easily with signal molecules including peptides and nucleic acids without side products providing great interest for non-viral gene therapy.

Intracellular trafficking and functional monitoring of miRNA delivery in glioblastoma using lipopolyplexes and the miRNA-ON RILES reporter system.

MicroRNA (miRNA) oligonucleotides therapeutics are potent and attractive drugs for cancer treatment, but the kinetics of their intracellular trafficking, RISC processing and interaction with their mRNA targets in the cells are still not well understood. Moreover, the absence of efficient carriers impairs their translation into the clinic. Here, we compare the kinetics of miRNA-133a activity after transfection of U87MG glioblastoma cells with either a home-made lipopolyplexes (LPRi) or with the RNAiMax transfection reagent. For this purpose, we combined miRNA intracellular trafficking studies by confocal microscopy with our previously described RILES miRNA-ON reporter system subcloned here in a lentivirus expression vector (LentiRILES) for longitudinal analysis of miRNA activity in transfected cells. Using the LentiRILES system, we report significant differences in terms of miRNA delivery kinetics performed by these two transfection regents. We decipher the mechanisms of miRNA delivery by LPRi and investigate the main steps of miRNA internalization and cytosolic processing. We demonstrate that LPRi preferentially uses caveolae-mediated endocytosis as the main internalization pathway, releases miRNA into the cytosol after the first 3 h of incubation, and addresses the cytosolic miRNAs to P-bodies, while a fraction of miRNAs are exported to the extracellular space through exosomes which were found fully capable to re-transfect the cells. We implanted the LentiRILES cells in the brain of mice and infused the tumours with LPRi.miRNA using the convection-enhanced delivery method. Bioluminescence imaging of the live mice revealed efficient delivery of miRNAs in glioblastoma tumours, attesting successful miRNA uptake, internalization and RISC activation in vivo. Overall, our study provides a comprehensive overview of miRNA intracellular trafficking and processing in a glioblastoma context and highlights the potential use of LPRi for miRNA-based therapy.

Co-delivery of NS1 and BMP2 mRNAs to murine pluripotent stem cells leads to enhanced BMP-2 expression and osteogenic differentiation.

Application of messenger RNA (mRNA) for bone regeneration is a promising alternative to DNA, recombinant proteins and peptides. However, exogenous in vitro transcribed mRNA (IVT mRNA) triggers innate immune response resulting in mRNA degradation and translation inhibition. Inspired by the ability of viral immune evasion proteins to inhibit host cell responses against viral RNA, we applied non-structural protein-1 (NS1) from Influenza A virus (A/Texas/36/1991) as an IVT mRNA enhancer. We evidenced a dose-dependent blocking of RNA sensors by NS1 expression. The co-delivery of NS1 mRNA with mRNA of reporter genes significantly increased the translation efficiency. Interestingly, unlike the use of nucleosides modification, NS1-mediated mRNA translation enhancement does not dependent to cell type. Dual delivery of NS1 mRNA and BMP-2 mRNA to murine pluripotent stem cells (C3H10T1/2), promoted osteogenic differentiation evidenced by enhanced expression of osteoblastic markers (e.g. alkaline phosphatase, type I collagen, osteopontin, and osteocalcin), and extracellular mineralization. Overall, these results support the adjuvant potentiality of NS1 for mRNA-based regenerative therapies. STATEMENT OF SIGNIFICANCE: mRNA therapy has the potential to improve the efficiency of nucleic acid based regenerative medicine. Up to now, the incorporation of expensive modified nucleotides is a common way to avoid IVT mRNA-induced detrimental immunogenicity. We here introduce co-delivery of Influenza virus immune evasion protein-NS1 coding mRNA as a strategy to suppress RNA sensors for maximizing IVT mRNA expression. An increased osteogenic commitment of pluripotent stem cells was observed after BMP2 mRNA and NS1 mRNA delivery. This study revealed how applying non-modified mRNA with NS1 could be a promising alternative as a therapeutic in bone regeneration.

Periodic Mesoporous Organosilica Nanoparticles with BOC Group, towards HIFU Responsive Agents.

Periodic Mesoporous Organosilica Nanoparticles (PMONPs) are nanoparticles of high interest for nanomedicine applications. These nanoparticles are not composed of silica (SiO2). They belong to hybrid organic-inorganic systems. We considered using these nanoparticles for CO2 release as a contrast agent for High Intensity Focused Ultrasounds (HIFU). Three molecules (P1-P3) possessing two to four triethoxysilyl groups were synthesized through click chemistry. These molecules possess a tert-butoxycarbonyl (BOC) group whose cleavage in water at 90-100 °C releases CO2. Bis(triethoxysilyl)ethylene E was mixed with the molecules Pn (or not for P3) at a proportion of 90/10 to 75/25, and the polymerization triggered by the sol-gel procedure led to PMONPs. PMONPs were characterized by different techniques, and nanorods of 200-300 nm were obtained. These nanorods were porous at a proportion of 90/10, but non-porous at 75/25. Alternatively, molecules P3 alone led to mesoporous nanoparticles of 100 nm diameter. The BOC group was stable, but it was cleaved at pH 1 in boiling water. Molecules possessing a BOC group were successfully used for the preparation of nanoparticles for CO2 release. The BOC group was stable and we did not observe release of CO2 under HIFU at lysosomal pH of 5.5. The pH needed to be adjusted to 1 in boiling water to cleave the BOC group. Nevertheless, the concept is interesting for HIFU theranostic agents.

Temperature-Sensitive Amphiphilic Non-Ionic Triblock Copolymers for Enhanced In Vivo Skeletal Muscle Transfection.

It is reported that low concentration of amphiphilic triblock copolymers of pMeOx-b-pTHF-b-pMeOx structure (TBCPs) improves gene expression in skeletal muscle upon intramuscular co-injection with plasmid DNA. Physicochemical studies carried out to understand the involved mechanism show that a phase transition of TBCPs under their unimer state is induced when the temperature is elevated from 25 to 37 °C, the body temperature. Several lines of evidences suggest that TBCP insertion in a lipid bilayer causes enough lipid bilayer destabilization and even pore formation, a phenomenon heightened during the phase transition of TBCPs. Interestingly, this property allows DNA translocation across the lipid bilayer model. Overall, the results indicate that TBCPs exhibiting a phase transition at the body temperature is promising to favor in vivo pDNA translocation in skeletal muscle cells for gene therapy applications.

Comparative binding and uptake of liposomes decorated with mannose oligosaccharides by cells expressing the mannose receptor or DC-SIGN.

Mannose Receptor (MR) and DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) are two mannose-specific targets for antigens carried by liposomes but DC-SIGN is more specific of DCs. Here, DC targeting is addressed by using DPPC/DOPE liposomes decorated with a series of diether lipids with a polar head of either a mannose (Man), tri-antenna of α-d-mannopyranoside (Tri-Man), [Manα1-3(Manα1-6)Man] (Man-tri), pseudo-Man4 (PMan4) or pseudo-Man5 (PMan5). Liposomes decorated with Man-Tri show the highest binding and internalization in cells expressing DC-SIGN and in human monocytes-derived DCs. Conversely, cells expressing MR bind and take up Tri-Man liposomes 3-fold higher than Man-tri liposomes. Comparatively, liposomes decorated with PMan4 and PMan5 do not show any advantages. Overall, the results indicate that liposomes decorated with Man-tri residues are more selective toward DCs than those with Tri-Man thanks to better recognition by DC-SIGN.

Gene transfer to skeletal muscle using hydrodynamic limb vein injection: current applications, hurdles and possible optimizations.

Hydrodynamic limb vein injection is an in vivo locoregional gene delivery method. It consists of administrating a large volume of solution containing nucleic acid constructs in a limb with both blood inflow and outflow temporarily blocked using a tourniquet. The fast, high pressure delivery allows the musculature of the whole limb to be reached. The skeletal muscle is a tissue of choice for a variety of gene transfer applications, including gene therapy for Duchenne muscular dystrophy or other myopathies, as well as for the production of antibodies or other proteins with broad therapeutic effects. Hydrodynamic limb vein delivery has been evaluated with success in a large range of animal models. It has also proven to be safe and well-tolerated in muscular dystrophy patients, thus supporting its translation to the clinic. However, some possible limitations may occur at different steps of the delivery process. Here, we have highlighted the interests, bottlenecks and potential improvements that could further optimize non-viral gene transfer following hydrodynamic limb vein injection.

Improved histidinylated lPEI polyplexes for skeletal muscle cells transfection.

Linear Polyethylenimine (lPEI) is an efficient cationic polymer for transfecting cells, both in vitro and in vivo, but poses concerns regarding cytotoxicity. Histidinylated lPEI (His-lPEI) exhibits also high transfection efficiency but lower cytotoxicity than lPEI. For the first time, we tested polyfection efficiency of polyplexes comprising both lPEI and His-lPEI. A series of pDNA polyplexes was prepared with mixtures of lPEI and His-lPEI and the amount of each polymer within His-lPEI/lPEI polyplexes was determined by flow cytometry. We show that His-lPEI/lPEI polyplexes exhibit properties similar to lPEI polyplexes in terms of size, morphology, assembly with pDNA, and polyplex stability while His-lPEI/lPEI polyplexes exhibit properties similar to His-lPEI polyplexes in terms of buffering capacity. Compared to polyplexes consisting only of lPEI or His-lPEI, the transfection profile reveals that His-lPEI/lPEI polyplexes containing 30% to 57% lPEI strongly increase polyfection efficiency of NIH3T3 fibroblasts and murine, as well as human skeletal muscle cell lines without cytotoxicity. Importantly, improved transfection of human dystrophin deficient skeletal muscle cell lines was obtained. These results indicate that His-lPEI/lPEI polyplexes are an improved non-viral vector for efficient transfection of dystrophin deficient skeletal muscle cell lines that should be tested on animals.