Non-viral Gene Delivery Methods Research Articles

Stereotaxic Surgery as a Method to Deliver Epigenetic Editing Constructs in Rodent Brain.

Modern neuroscience research is increasingly discovering that alterations in epigenetic states within key brain cells is correlated with brain diseases. These epigenetic alterations may include changes in histone post-translational modifications and/or DNA modifications, all of which affect transcription and other gene expression programs within the brain cells that comprise central brain regions. However, the exact causal contribution of these epigenome changes to brain disease cannot be elucidated in the absence of direct in vivo manipulations in the implicated brain areas. Combining the design and creation of epigenetic editing constructs, gene delivery strategies, and stereotaxic surgery enables neuroscience researchers to target and manipulate the epigenetic state of the brain cells of laboratory rodents in a locus-specific manner and test its causal contribution to disease-related pathology and behaviors. Here, we describe the surgical protocol utilized by our group and others, which is optimized for herpes simplex virus delivery into the mouse brain, although the protocol outlined herein could be applied for delivery of adeno-associated viruses, lentiviruses, or nonviral gene-delivery methods in both mice and rats. The method allows for the overexpression of engineered DNA-binding proteins for direct and targeted epigenome editing in rodent brain with excellent spatiotemporal control. Nearly any brain region of interest can be targeted in rodents at every stage of postnatal life. Owing to the versatility, reproducibility, and utility of this technique, it is an important method for any laboratory interested in studying the cellular, circuit, and behavioral consequences of manipulating the brain epigenome in laboratory rodents.

An Overview of Gene Editing Modalities and Related Non-clinical Testing Considerations.

Gene therapy has become an important modality for a wide range of therapeutic indications with a rapid increase in the number of therapeutic candidates being developed in this field. Understanding the molecular biology underlying the gene therapy is often critical to develop appropriate safety assessment strategies. We aimed to discuss some of the commonly used gene therapy modalities and common preclinical toxicology testing considerations when developing gene therapies. Non-viral gene delivery methods such as electroporation, microinjection, peptide nanoparticles and lipid nanoparticles are deployed as innovative molecular molecular construct which are included in the design of novel gene therapies and the associated molecular biology mechanisms have become relevant knowledge to non-clinical toxicology. Viral gene delivery methodologies including Adenovirus vectors, Adeno-Associated virus vectors and Lentivirus gene therapy vectors have also advanced considerably across numerous therapeutic areas, raising unique non-clinical toxicology and immunological considerations. General toxicology, biodistribution and tumorigenicity are the pillars of non-clinical safety testing in gene therapies. Evaluating the tumorigenicity potential of a gene editing therapy often leverages molecular pathology while some translational challenges remain. Toxicology study design is entering a new era where science-driven customized approaches and program specific considerations have become the norm.

The Progress of Non-Viral Materials and Methods for Gene Delivery to Skeletal Muscle.

Since Jon A. Wolff found skeletal muscle cells being able to express foreign genes and Russell J. Mumper increased the gene transfection efficiency into the myocytes by adding polymers, skeletal muscles have become a potential gene delivery and expression target. Different methods have been developing to deliver transgene into skeletal muscles. Among them, viral vectors may achieve potent gene delivery efficiency. However, the potential for triggering biosafety risks limited their clinical applications. Therefore, non-viral biomaterial-mediated methods with reliable biocompatibility are promising tools for intramuscular gene delivery in situ. In recent years, a series of advanced non-viral gene delivery materials and related methods have been reported, such as polymers, liposomes, cell penetrating peptides, as well as physical delivery methods. In this review, we summarized the research progresses and challenges in non-viral intramuscular gene delivery materials and related methods, focusing on the achievements and future directions of polymers.

A BAFF ligand-based CAR-T cell targeting three receptors and multiple B cell cancers

B cell-activating factor (BAFF) binds the three receptors BAFF-R, BCMA, and TACI, predominantly expressed on mature B cells. Almost all B cell cancers are reported to express at least one of these receptors. Here we develop a BAFF ligand-based chimeric antigen receptor (CAR) and generate BAFF CAR-T cells using a non-viral gene delivery method. We show that BAFF CAR-T cells bind specifically to each of the three BAFF receptors and are effective at killing multiple B cell cancers, including mantle cell lymphoma (MCL), multiple myeloma (MM), and acute lymphoblastic leukemia (ALL), in vitro and in vivo using different xenograft models. Co-culture of BAFF CAR-T cells with these tumor cells results in induction of activation marker CD69, degranulation marker CD107a, and multiple proinflammatory cytokines. In summary, we report a ligand-based BAFF CAR-T capable of binding three different receptors, minimizing the potential for antigen escape in the treatment of B cell cancers.

Effects of Gene Delivery Approaches on Differentiation Potential and Gene Function of Mesenchymal Stem Cells.

Introduction of a gene to mesenchymal stem cells (MSCs) is a well-known strategy to purposely manipulate the cell fate and further enhance therapeutic performance in cell-based therapy. Viral and chemical approaches for gene delivery interfere with differentiation potential. Although microinjection as a physical delivery method is commonly used for transfection, its influence on MSC cell fate is not fully understood. The current study aimed to evaluate the effects of four nonviral gene delivery methods on stem cell multi-potency. The four delivery methods are robotic microinjection, polyethylenimine (PEI), cationic liposome (cLipo), and calcium phosphate nanoparticles (CaP). Among the four methods, microinjection has exhibited the highest transfection efficiency of ∼60%, while the three others showed lower efficiency of 10-25%. Robotic microinjection preserved fibroblast-like cell morphology, stress fibre intactness, and mature focal adhesion complex, while PEI caused severe cytotoxicity. No marked differentiation bias was observed after microinjection and cLipo treatment. By contrast, CaP-treated MSCs exhibited excessive osteogenesis, while PEI-treated MSCs showed excessive adipogenesis. Robotic microinjection system was used to inject the CRISPR/Cas9-encoding plasmid to knock out PPARγ gene in MSCs, and the robotic microinjection did not interfere with PPARγ function in differentiation commitment. Meanwhile, the bias in osteo-adipogenic differentiation exhibited in CaP and PEI-treated MSCs after PPARγ knockout via chemical carriers. Our results indicate that gene delivery vehicles variously disturb MSCs differentiation and interfere with exogenous gene function. Our findings further suggest that robotic microinjection offers a promise of generating genetically modified MSCs without disrupting stem cell multi-potency and therapeutic gene function.

Impact of photobiomodulation using four diode laser wavelengths of on cationic liposome gene transfection into pre-osteoblast cells

Gene therapy can be an effective treatment modality for some severe genetic diseases. Despite efforts to improve their performance, non-viral gene delivery methods remain inefficient and costly. As an alternative to viral vectors, cationic liposomes have a good safety profile and low immunogenicity, but relatively low transfection efficiency. They may also be toxic to cells at high concentrations. Given these challenges, the present study explored the impact of photobiomodulation (PBM) on cationic liposome plasmid DNA transfection in terms of its efficiency and toxicity, using Lipofectamine 2000 to carry green fluorescent protein (GFP) encoding plasmid DNA, with the pre-osteoblast MC3T3-E1 cell line as the target. Cultures were irradiated using diode lasers (445, 685, 810, or 970 nm) at 200 mW using pulsed mode (50 Hz), with a power density of 104.64 mW/cm2, and irradiance from 6 to 18 joules. To determine transfection efficiency, expression of GFP was assessed using confocal laser scanning microscopy and flow cytometry. Cell viability was evaluated using the MTT assay. PBM using 810 nm and 970 nm lasers significantly enhanced transfection efficiency for GFP, indicating more efficient uptake of plasmid DNA. Conversely, laser irradiation at 445 nm and 685 nm wavelengths reduced the GFP transfection efficiency. Treatment using 685, 810, and 970 nm lasers at 12 J maintained cell viability and prevented toxicity of cationic liposomes. Overall, these findings support the concept that PBM using near infrared laser wavelengths can enhance transfection efficiency and support cell viability when cationic liposomes are used as the vector in gene therapy.

CRISPR-Cas9 based genome editing for defective gene correction in humans and other mammals.

Clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR/Cas9), derived from bacterial and archean immune systems, has received much attention from the scientific community as a powerful, targeted gene editing tool. The CRISPR/Cas9 system enables a simple, relatively effortless and highly specific gene targeting strategy through temporary or permanent genome regulation or editing. This endonuclease has enabled gene correction by taking advantage of the endogenous homology directed repair (HDR) pathway to successfully target and correct disease-causing gene mutations. Numerous studies using CRISPR support the promise of efficient and simple genome manipulation, and the technique has been validated in in vivo and in vitro experiments, indicating its potential for efficient gene correction at any genomic loci. In this chapter, we detailed various strategies related to gene editing using the CRISPR/Cas9 system. We also outlined strategies to improve the efficiency of gene correction via the HDR pathway and to improve viral and non-viral mediated gene delivery methods, with an emphasis on their therapeutic potential for correcting genetic disorder in humans and other mammals.

Non-viral Gene Delivery Methods for Bone and Joints

Viral carrier transport efficiency of gene delivery is high, depending on the type of vector. However, viral delivery poses significant safety concerns such as inefficient/unpredictable reprogramming outcomes, genomic integration, as well as unwarranted immune responses and toxicity. Thus, non-viral gene delivery methods are more feasible for translation as these allow safer delivery of genes and can modulate gene expression transiently both in vivo, ex vivo, and in vitro. Based on current studies, the efficiency of these technologies appears to be more limited, but they are appealing for clinical translation. This review presents a summary of recent advancements in orthopedics, where primarily bone and joints from the musculoskeletal apparatus were targeted. In connective tissues, which are known to have a poor healing capacity, and have a relatively low cell-density, i.e., articular cartilage, bone, and the intervertebral disk (IVD) several approaches have recently been undertaken. We provide a brief overview of the existing technologies, using nano-spheres/engineered vesicles, lipofection, and in vivo electroporation. Here, delivery for microRNA (miRNA), and silencing RNA (siRNA) and DNA plasmids will be discussed. Recent studies will be summarized that aimed to improve regeneration of these tissues, involving the delivery of bone morphogenic proteins (BMPs), such as BMP2 for improvement of bone healing. For articular cartilage/osteochondral junction, non-viral methods concentrate on targeted delivery to chondrocytes or MSCs for tissue engineering-based approaches. For the IVD, growth factors such as GDF5 or GDF6 or developmental transcription factors such as Brachyury or FOXF1 seem to be of high clinical interest. However, the most efficient method of gene transfer is still elusive, as several preclinical studies have reported many different non-viral methods and clinical translation of these techniques still needs to be validated. Here we discuss the non-viral methods applied for bone and joint and propose methods that can be promising in clinical use.

Optimization of electroporation and other non-viral gene delivery strategies for T cells.

CAR-T therapy is a particularly effective treatment for some types of cancer that uses retroviruses to deliver the gene for a chimeric antigen receptor (CAR) to a patient's T cells ex vivo. The CAR enables the T cells to bind and eradicate cells with a specific surface marker (e.g., CD19+ B cells) after they are transfused back into the patient. This treatment was proven to be particularly effective in treating non-Hodgkin's lymphoma (NHL) and acute lymphoblastic leukemia (ALL), but the current CAR-T cell manufacturing process has a few significant drawbacks. For example, while lentiviral and gammaretroviral transduction are both relatively effective, the process of producing viral vectors is time-consuming and costly. Additionally, patients must undergo follow up appointments for several years to monitor them for any unanticipated side effects associated with the virus. Therefore, several studies have endeavored to find alternative non-viral gene delivery methods that are less expensive, more precise, simple, and safe. This review focuses on the current state of the most promising non-viral gene delivery techniques, including electroporation and transfection with cationic polymers or lipids.

The challenging nature of primary T lymphocytes for transfection: Effect of protamine sulfate on the transfection efficiency of chemical transfection reagents.

Background and purpose:The optimization of an effective non-viral gene delivery method for genetic manipulation of primary human T cells has been a major challenge in immunotherapy researches. Due to the poor transfection efficiency of conventional methods in T cells, there has been an effort to increase the transfection rate in these cells. Protamine is an FDA-approved compound with a documented safety profile that enhances DNA condensation for gene delivery.Experimental approach:In this study, the effect of protamine sulfate on the transfection efficiency of standard transfection reagents, was evaluated to transfect primary human T cells. In this regard, pre-condensation of DNA was applied using protamine, and the value of the zeta potential of DNA/protamine/cargo complexes was determined. T cells were transfected with DNA/protamine/cargo complexes. The transfection efficiency rate was evaluated by flow cytometry. Also, the green fluorescent protein expression level and cytotoxicity of each complex were identified using real-time polymerase chain reaction and MTT assay, respectively.Findings/Results:Our results demonstrated that protamine efficiently increases the positive charge of DNA/cargo complex without any cytotoxic effect on the primary human T cells. We observed that the transfection efficiency in DNA/protamine/ Lipofectamine® 2000 and DNA/protamine/TurboFect™ was 87.2% and 78.9%, respectively, while transfection of T cells by Lipofectamine® 2000 and TurboFect™ would not result in sufficient transfection.Conclusion and implications:Protamine sulfate enhanced the transfection rate of T cells; and could be a promising non-viral gene delivery method to achieve a safe, rapid, cost-effective, and efficient system which will be further applied in gene therapy and T cells manipulation methods.

Plasmid-Mediated Gene Therapy in Mouse Models of Limb Girdle Muscular Dystrophy

We delivered plasmid DNA encoding therapeutic genes to the muscles of mouse models of limb girdle muscular dystrophy (LGMD) 2A, 2B, and 2D, deficient in calpain3, dysferlin, and alpha-sarcoglycan, respectively. We also delivered the human follistatin gene, which has the potential to increase therapeutic benefit. After intramuscular injection of DNA, electroporation was applied to enhance delivery to muscle fibers. When plasmids encoding the human calpain3 or dysferlin cDNA sequences were injected into quadriceps muscles of LGMD2A and LGMD2B mouse models, respectively, in 3-month studies, robust levels of calpain3 and dysferlin proteins were detected. We observed a statistically significant decrease in Evans blue dye penetration in LGMD2B mouse muscles after delivery of the dysferlin gene, consistent with repair of the muscle membrane defect in these mice. The therapeutic value of delivery of the genes for alpha-sarcoglycan and follistatin was documented by significant drops in Evans blue dye penetration in gastrocnemius muscles of LGMD2D mice. These results indicated for the first time that a combined gene therapy involving both alpha-sarcoglycan and follistatin would be valuable for LGMD2D patients. We suggest that this non-viral gene delivery method should be explored for its translational potential in patients.

Non-viral gene delivery for cancer immunotherapy.

Recent decades have witnessed the revolutionary development of cancer immunotherapies, which boost cancer-specific immune responses for long-term tumor regression. However, immunotherapy still has limitations, including off-target side effects, long processing times and limited patient responses. These disadvantages of current immunotherapy are being addressed by improving our understanding of the immune system, as well as by establishing combinational approaches. Advanced biomaterials and gene delivery systems overcome some of these delivery issues, harnessing adverse effects and amplifying immunomodulatory effects, and are superior to standard formulations with respect to eliciting antitumor immunity. Nucleic acid-based nanostructures have diverse functions, ranging from gene expression and gene regulation to pro-inflammatory effects, as well as the ability to specifically bind different molecules. A brief overview is provided of the recent advances in the non-viral gene delivery methods that are being used to activate cancer-specific immune responses. Furthermore, the tumor microenvironment-responsive synergistic strategies that modulate the immune response by targeting various signaling pathways are discussed. Nanoparticle-based non-viral gene delivery strategies have great potential to be implemented in the clinic for cancer immunotherapy.

Glycopolycation-DNA Polyplex Formulation N/P Ratio Affects Stability, Hemocompatibility, and in Vivo Biodistribution.

Genome editing therapies hold great promise for the cure of monogenic and other diseases; however, the application of nonviral gene delivery methods is limited by both a lack of fundamental knowledge of interactions of the gene-carrier in complex animals and biocompatibility. Herein, we characterize nonviral gene delivery vehicle formulations that are based on diblock polycations containing a hydrophilic and neutral glucose block chain extended with cationic secondary amines of three lengths, poly(methacrylamido glucopyranose- block-2-methylaminoethyl methacrylate) [P(MAG- b-MAEMt)-1, -2, -3]. These polymers were formulated with plasmid DNA to prepare polyelectrolyte complexes (polyplexes). In addition, two controls, P(EG- b-MAEMt) and P(MAEMt), were synthesized, formulated into polyplexes and the ex vivo hemocompatibility, or blood compatibility, and in vivo biodistribution of the formulations were compared to the glycopolymers. While both polymer structure and N/P (amine to phosphate) ratio were important factors affecting hemocompatibility, N/P ratio played a stronger role in determining polyplex biodistribution. P(EG- b-MAEMt) and P(MAEMt) lysed red blood cells at both high and low N/P formulations while P(MAG- b-MAEMt) did not significantly lyse cells at either formulation at short and medium polymer lengths. Conversely, P(MAG- b-MAEMt) did not affect coagulation at N/P = 5, but significantly delayed coagulation at N/P = 15. P(EG- b-MAEMt) and P(MAEMt) did not affect coagulation at either formulation. After polymer and pDNA cargo distribution was observed in vivo, P(EG- b-MAEMt) N/P = 5 and P(MAG- b-MAEMt) N/P = 5 both dissociated and deposited polymer in the liver, while pDNA cargo from P(MAG- b-MAEMt) N/P = 15 was found in the liver, lungs, and spleen. The contrast between P(MAG- b-MAEMt) at N/P = 5 and 15 demonstrates that polyplex stability in the blood can be improved with N/P ratio and potentially aid polyplex biodistribution through simply varying the formulation ratios.

Successful genetic modification of porcine spermatogonial stem cells via an electrically responsive Au nanowire injector

Transgenic pigs are quite useful in many biomedical fields, such as xenotransplantation research and the production of biopharmaceutical materials. The genetic transformation of porcine spermatogonial stem cells (pSSCs) followed by differentiation into mature spermatozoa enables the effective production of transgenic pigs. Improving the transfection efficiency of pSSCs, however, has been much desired. Herein, we report the efficient genetic modification of pSSCs by using an electrically responsive Au nanowire injector (E-R Au NWI). This is the first study that shows an exogenous gene is directly delivered into the nucleus of a pSSC by using a 1-dimensional nanomaterial and then successfully expressed to produce a protein. The E-R Au NWI interfaced noninvasively with the nucleus of the pSSC, and the pEGFP-N1 plasmid was delivered by the application of an electrical stimulus without cell damage. Compared to the results of conventional nonviral vector-based gene delivery methods such as jetPEI, Lipofectamine, and electroporation, the E-R Au NWI-based method improved the pSSC transfection efficiency by at least 6.7-fold and even up to 46.7-fold. Furthermore, we successfully obtained transgenic pSSCs containing the human bone morphogenetic protein 2 gene by using E-R Au NWIs. This result suggests that the E-R Au NWI enables the efficient genetic modification of pSSCs and can be employed to produce diverse kinds of transgenic pigs.

Safe and efficient novel approach for non-invasive gene electrotransfer to skin

Gene transfer into cells or tissue by application of electric pulses (i.e. gene electrotransfer (GET)) is a non-viral gene delivery method that is becoming increasingly attractive for clinical applications. In order to make GET progress to wide clinical usage its efficacy needs to be improved and the safety of the method has to be confirmed. Therefore, the aim of our study was to increase GET efficacy in skin, by optimizing electric pulse parameters and the design of electrodes. We evaluated the safety of our novel approach by assaying the thermal stress effect of GET conditions and the biodistribution of a cytokine expressing plasmid. Transfection efficacy of different pulse parameters was determined using two reporter genes encoding for the green fluorescent protein (GFP) and the tdTomato fluorescent protein, respectively. GET was performed using non-invasive contact electrodes immediately after intradermal injection of plasmid DNA into mouse skin. Fluorescence imaging of transfected skin showed that a sophistication in the pulse parameters could be selected to get greater transfection efficacy in comparison to the standard ones. Delivery of electric pulses only mildly induced expression of the heat shock protein Hsp70 in a luminescent reporting transgenic mouse model, demonstrating that there were no drastic stress effects. The plasmid was not detected in other organs and was found only at the site of treatment for a limited period of time. In conclusion, we set up a novel approach for GET combining new electric field parameters with high voltage short pulses and medium voltage long pulses using contact electrodes, to obtain a high expression of both fluorescent reporter and therapeutic genes while showing full safety in living animals.

Nanosecond range electric pulse application as a non-viral gene delivery method: proof of concept

Current electrotransfection protocols are well-established for decades and, as a rule, employ long micro-millisecond range electric field pulses to facilitate DNA transfer while application of nanosecond range pulses is limited. The purpose of this paper is to show that the transfection using ultrashort pulses is possible by regulating the pulse repetition frequency. We have used 200 ns pulses (10–18 kV/cm) in bursts of ten with varied repetition frequency (1 Hz–1 MHz). The Chinese Hamster Ovary (CHO) cells were used as a cell model. Experiments were performed using green fluorescent protein (GFP) and luciferase (LUC) coding plasmids. Transfection expression levels were evaluated using flow cytometry or luminometer. It was shown that with the increase of frequency from 100 kHz to 1 MHz, the transfection expression levels increased up to 17% with minimal decrease in cell viability. The LUC coding plasmid was transferred more efficiently using high frequency bursts compared to single pulses of equivalent energy. The first proof of concept for frequency-controlled nanosecond electrotransfection was shown, which can find application as a new non-viral gene delivery method.

Ternary complex of plasmid DNA with NLS-Mu-Mu protein and cationic niosome for biocompatible and efficient gene delivery: a comparative study with protamine and lipofectamine

Non-viral gene delivery methods are considered due to safety and simplicity in human gene therapy. Since the use of cationic peptide and niosome represent a promising approach for gene delivery purposes we used recombinant fusion protein and cationic niosome as a gene carrier. A multi-domain fusion protein including nuclear localization motif (NLS) and two DNA-binding (Mu) domains, namely NLS-Mu-Mu (NMM) has been designed, cloned and expressed in E. coli DE3 strain. Afterward, the interested protein was purified by affinity chromatography. Binary vectors based on protein/DNA and ternary vectors based on protein/DNA/niosome were prepared. Protamine was used as a control. DNA condensing properties of NMM and protamine were evaluated by various experiments. Furthermore, we examined cytotoxicity, hemolysis and transfection potential of the binary and ternary complexes in HEK293T and MCF-7 cell lines. Protamine and Lipofectamine™2000 were used as positive controls, correspondingly. The recombinant NMM was expressed and purified successfully and DNA was condensed efficiently at charge ratios that were not harmful to cells. Peptidoplexes showed transfection efficiency (TE) but ternary complexes had higher TE. Additionally, NMM ternary complex was more efficient compared to protamine ternary vectors. Our results showed that niosomal ternary vector of NMM is a promising non-viral gene carrier to achieve an effective and safe carrier system for gene therapy.

Direct Incorporation of Functional Peptides into M-DNA through Ligand-to-Metal Charge Transfer.

Conventional nonviral gene delivery methods suffer from the toxicity of the cationic nature of polymeric carriers. There is a significant need for a new method of gene delivery that overcomes the limitations and allows targeted gene delivery. In this study, we have developed a new method to incorporate functional peptides into DNA without the need for chemical conjugations by utilizing a ligand-to-metal charge transfer (LMCT) transition, which occurs between divalent metal ions and the sulfhydryl group in cysteine. To apply the LMCT transition to the incorporation of cysteine-containing targeting peptides into DNA, divalent metal ions must be first introduced to DNA. Zn2+ ions spontaneously intercalate into the DNA base pairs in the pH range of 7.0-8.5, resulting in the conversion of normal B-DNA to metal-bound DNA (M-DNA). We found that the Zn2+ ions present in M-DNA could interact with the sulfhydryl groups in cysteines of targeting peptides through the LMCT transition, and the M-DNA/peptide complex could specifically transfect the target cells.

Efficient and non-toxic gene delivery by anionic lipoplexes based on polyprenyl ammonium salts and their effects on cell physiology.

One of the major challenges limiting the development of gene therapy is an absence of efficient and safe gene carriers. Among the nonviral gene delivery methods, lipofection is considered as one of the most promising. In the present study, a set of cationic polyprenyl derivatives [trimethylpolyprenylammonium iodides (PTAI)] with different lengths of polyprenyl chains (from 7, 8 and 11 to 15 isoprene units) was suggested as a component of efficient DNA vehicles. Optimization studies were conducted for PTAI in combination with co-lipid dioleoylphosphatidylethanolamine on DU145 human prostate cancer cells using: size and zeta potential measurements, confocal microscopy, the fluorescein diacetate/ethidium bromide test, cell counting, time-lapse monitoring of cell movement, gap junctional intercellular coupling analysis, antimicrobial activity assay and a red blood cell hemolysis test. The results obtained show that the lipofecting activity of PTAI allows effective transfection of plasmid DNA complexed in negatively-charged lipoplexes of 200-500nm size into cells without significant side effects on cell physiology (viability, proliferation, morphology, migration and gap junctional intercellular coupling). Moreover, PTAI-based vehicles exhibit a potent bactericidal activity against Staphylococcus aureus and Escherichia coli. The developed anionic lipoplexes are safe towards human red blood cell membranes, which are not disrupted in their presence. The developed carriers constitute a group of promising lipofecting agents of a new type that can be utilized as effective lipofecting agents in vitro and they are also an encouraging basis for in vivo applications.

Combined Local Pulmonary and Systemic Delivery of AT2R Gene by Modified TAT Peptide Nanoparticles Attenuates Both Murine and Human Lung Carcinoma Xenografts in Mice

To evaluate the potential of cell-penetrating peptide-based delivery of apoptosis-inducer gene in cancer therapy, a modified HIV-1 TAT peptide (dimerized TAT peptide, dTAT) was studied. The dTAT and plasmid DNA (pDNA) complexes (dTAT-pDNA) were condensed using calcium chloride (dTAT-pDNA-Ca2+). This simple nonviral formulation approach showed high levels of gene expression in vitro without any cytotoxicity. In mouse studies, a single intratracheal (IT) aerosol spray or 2 intravenous (IV) injections of the dTAT, apoptosis-inducer gene, angiotensin II type 2 receptor (AT2R), and Ca2+ complexes (dTAT-pAT2R-Ca2+) significantly attenuated the acutely growing mouse Lewis lung carcinoma allografts in mouse lungs. Furthermore, single IT (p = 0.054) and the combination of IT and IV (p < 0.05) administrations of dTAT-pAT2R-Ca2+ markedly attenuated slowly growing and relatively large-sized H358 human bronchioloalveolar carcinoma xenografts in mouse lungs. These results indicate that the dTAT-pDNA-Ca2+ effectively delivered the gene to cancer cells by either IT or IV administration although the local pulmonary delivery of the dTAT-pAT2R-Ca2+ showed more effective growth inhibition of orthotopic lung cancer grafts. Thus, the present study offers preclinical proof of concept that a dTAT-based nonviral gene delivery method via IT administration may be an effective lung cancer gene therapy.