Non-viral Gene Therapy Research Articles

Nonviral Gene Therapy for Cancer: A Review.

Although the development of effective viral vectors put gene therapy on the road to commercialization, nonviral vectors show promise for practical use because of their relative safety and lower cost. A significant barrier to the use of nonviral vectors, however, is that they have not yet proven effective. This apparent lack of interest can be attributed to the problem of the low gene transfer efficiency associated with nonviral vectors. The efficiency of gene transfer via nonviral vectors has been reported to be 1/10th to 1/1000th that of viral vectors. Despite the fact that new gene transfer methods and nonviral vectors have been developed, no significant improvements in gene transfer efficiency have been achieved. Nevertheless, some notable progress has been made. In this review, we discuss studies that report good results using nonviral vectors in vivo in animal models, with a particular focus on studies aimed at in vivo gene therapy to treat cancer, as this disease has attracted the interest of researchers developing nonviral vectors. We describe the conditions in which nonviral vectors work more efficiently for gene therapy and discuss how the goals might differ for nonviral versus viral vector development and use.

Intra-articular nonviral gene therapy in mucopolysaccharidosis I mice

Mucopolysaccharidosis type I (MPS I) is caused by the lysosomal accumulation of glycosaminoglycans (GAGs) due to the deficiency of the enzyme alpha-L-iduronidase (IDUA). Currently available treatments may improve several clinical manifestations, but they have limited effects on joint disease, resulting in persistent orthopedic complications and impaired mobility. Thus, this study aimed to perform an intra-articular administration of cationic nanoemulsions complexed with the plasmid encoding for the IDUA protein (pIDUA) targeting MPS I gene therapy for the synovial joints. Formulations composed of DOPE, DOTAP, MCT (NE), and DSPE-PEG (NE-PEG) were prepared by high-pressure homogenization, and the pIDUA plasmid was associated by adsorption onto the surface of nanoemulsions (pIDUA/NE or pIDUA/NE-PEG). The physicochemical characterization showed that the presence of DSPE-PEG in pIDUA/NE-PEG formulations led to small and highly stable droplets even when incubated with simulated synovial fluid (SSF), when compared to the non-pegylated complexes (pIDUA/NE). Uptake by fibroblast-like synoviocytes (FLS) was demonstrated, and high cell viability (70%) in addition with increased IDUA activity (2.5% of normal) were observed after incubation with pIDUA/NE-PEG. The intra-articular injection of pIDUA/NE-PEG complexes in MPS I mice showed that the complexes were localized in the joints, were able to transfect synovial cells, and thus promoted an increase in IDUA activity and expression in the synovial fluid, with no significant activity in other tissues (kidney, liver, lung, and spleen). The overall results demonstrated a contained, safe, tolerable, and effective in situ approach of nonviral intra-articular gene therapy targeting the reduction or prevention of the debilitating orthopedic complications of MPS I disorder.

BMP-2 gene transfection of bone marrow stromal cells to induce osteoblastic differentiation in a rat calvarial defect model

Gene therapy for bone tissue engineering has been widely developed. Recently, non-viral DNA-based gene therapy has been reported to be a safer and more efficient method of delivering DNA into target cells. We used a non-viral gene transfection reagent to delivery bone morphogenetic protein-2 (BMP-2) gene into bone marrow stromal cells (BMSCs). Primary BMSCs were isolated from rat femurs and transfected with BMP-2 plasmids. The transfection rate was analyzed using flow cytometry. The concentration of BMP-2 protein was quantified using an enzyme-linked immunosorbent assay. Levels of osteopontin and osteocalcin were measured to evaluate osteogenic differentiation. In vivo, we designed a critical-size calvarial defect rat model to study new bone regeneration, using Matrigel as a scaffold to carry BMP-2-transfected bone marrow stromal cells into the defect site. New bone formation was assessed by micro-computed tomography, X-ray, immunohistochemical staining and histomophometry. The transfection rate after 72 h was 31.5%. The BMP-2 protein level as well as osteopontin and osteocalcin expressions were higher in the experimental group (transfected with BMP-2) than the control group (transfected with green fluorescent protein, GFP). The in vivo study suggested that bone healing occurred 12 weeks after scaffold implantation. In addition, BMP-2-transfected bone marrow stromal cells provided better osteogenic differentiation than primary bone marrow stromal cells. Our findings suggest that non-viral gene therapy may be useful in bone tissue engineering. SignificanceThe study has clinical implications for the wider use of BMP-2-transfected BMSCs for cell-based transplantation therapy in bone regeneration.

Bacterial tRNase-Based Gene Therapy with Poly(β-Amino Ester) Nanoparticles for Suppressing Melanoma Tumor Growth and Relapse.

Here, a novel anticancer gene therapy with a bacterial tRNase gene, colicin D or virulence associated protein C (VapC), is suggested using biodegradable polymeric nanoparticles, such as poly(β-amino esters) (PBAEs) as carriers. These genes are meticulously selected, aiming at inhibiting translation in the recipients by hydrolyzing specific tRNA species. In terms of nanoparticles, out of 9 PBAE formulations, a leading polymer, (polyethylene oxide)4 -bis-amine end-capped poly(1,4-butanediol diacrylate-co-5-amino-1-pentanol) (B4S5E5), is identified that displays higher gene delivery efficacy to cancer cells compared with the leading commercial reagent Lipofectamine 2000. Interestingly, the B4S5E5 PBAE nanoparticles complexed with colicin D or VapC plasmid DNA induce significant toxicity highly specific to cancer cells by triggering apoptosis. In contrast, the PBAE nanoparticles do not induce these cytotoxic effects in noncancerous cells. In a mouse melanoma model of grafted murine B16-F10 cells, it is demonstrated that treatment with PBAE nanoparticles complexed with these tRNase genes significantly reduces tumor growth rate and delays tumor relapse. Moreover, increased stability of PBAE by PEGylation further enhances the therapeutic effect of tRNase gene treatment and improves survival of animals. This study highlights a nonviral gene therapy that is highly promising for the treatment of cancer.

PO-016 Inhibition of cell proliferation by anti-epidermal growth factor receptor (EGFR) aptamer conjugated chitosan/siRNA nanoparticles

Introduction Gene silencing mediated by small interfering RNA (siRNA) has been widely investigated as a potential therapeutic approach. Its use, however, is hampered by its rapid degradation and poor cellular uptake into cells. Therefore, the success will depend on the design of effective systems able to selectively and efficiently deliver siRNA to target cells/organs. Our strategy relies on the use of a biocompatible biopolymer (chitosan) as carrier of siRNA coupled with specific anti-EGFR aptamers for cell targeting which is overexpressed in various cancer cells. Finally, poly (ethylene glycol) (PEG) and diethylaminoethyl (DEAE) will be covalently-linked with chitosan, in order to improve blood residency and transfection efficiency. The selected siRNA will be directed to silence receptor activator of nuclear factor-kB ligand (RANKL). Its levels are elevated in numerous cancers. Blockage of EGFR by aptamer and knockdown of RANKL by siRNA inhibit cancer cell proliferation in vitro. Material and methods Synthesise and characterise chitosan conjugates: poly (ethylene glycol) (PEG), diethylaminoethyl (DEAE), and anti-EGFR-aptamer are covalently-linked with chitosan. Synthetize, purify and characterise DEAE/PEG/anti-EGFR-aptamer-chitosan/siRNA nanoparticles Optimise the nanoformulations (adjusting polymeric and charge ratios) through tests in vitro including transfection efficacy and cell proliferation assays in different cancer cell lines. Results and discussions Nanoparticles were produced on the basis of our previous results. Particle size and zeta potential were measured by Zetasizer Nano ZS90 (Malvern Instruments Ltd., Malvern, UK). The sizes of synthesised nanoparticles were around 259±3 nm for Chitosan-DEAE15/siRNA with a zeta potential of +28.3±0.8 mV. The average cell viability of free siRNA or nanoparticle-treated cells was 89%–97% compared to nonrelated cells. The results showed that anti-EGFR-aptamer-chitosan/siRNA nanoparticles had a dose-dependent inhibition of cell proliferation. These nanoparticles had a significant inhibition effect of RANKL mRNA expression (RT-PCR assay. Conclusion Conventional cancer treatments such as chemotherapy have severe side toxicity on both tumour and host cells. Biological targeted agents such as monoclonal antibodies are available, but costly. EGFR and RANKL are two of major targets for drug development for cancer treatment. Non-viral gene therapy involved aptamer-EGFR and siRNA-RANKL is a promising therapy strategy. The supportIng grant was from MESI-Quebec (PSR-SIIRI, 2017–2020).

The proton sponge hypothesis: Fable or fact?

In non-viral gene therapy, cationic polymers and lipids are frequently used to encapsulate macromolecular therapeutics into nanoparticles. During their journey to deliver the cargo to the intended intracellular target, many biological barriers need to be overcome. One of the major bottlenecks for efficient transfection is the endosomal barrier since nanoparticles often remain entrapped inside endosomes and are trafficked towards the lysosomes where the cargo is degraded. For cationic polymers, the proton sponge hypothesis was introduced in the late ‘90s as a way to explain their endosomal escape properties. However, to date, no consensus has been reached in the scientific community about the validity of this hypothesis due to many contradictory reports. Here we review the sometimes conflicting reports that have been published on the proton sponge hypothesis. We also discuss membrane destabilization and polymer swelling as additional factors that might influence endosomal escape of polyplexes. Based on the key publications on this subject, we aim to launch a consensus on the role of the proton sponge hypothesis in endosomal escape.

Targeted Nanoparticle‐Mediated Gene Therapy Mimics Oncolytic Virus for Effective Melanoma Treatment

AbstractOncolytic virus has potential applications in cancer therapy. However, its clinical application is restricted by the virus‐associated biosafety issues. Here, inspired by the key role of vesicular stomatitis virus matrix protein (VSVMP) in the oncolytic vesicular stomatitis virus (VSV) induced apoptosis, a targeted nanoparticle‐delivered neutral VSVMP gene formulation is designed to act like the VSV for cancer therapy. This VSVMP formulation consists of a CRGDKGPDC peptide modified hybrid monomethoxy poly (ethylene glycol)‐poly(d,l‐lactide) nanoparticles complexed with VSVMP plasmid, having good blood compatibility and tumor targeting ability. The transfection efficiency is as high as that of VSV. After intravenous administration, the VSVMP formulation can efficiently target the tumor, significantly inhibit the melanoma growth and metastasis, prolong the survival time of tumor‐bearing mice, and does not cause obvious systemic toxicity. The anticancer mechanisms involve apoptosis induction, angiogenesis inhibition and some virus‐associated signal pathways activation. This work demonstrates a VSV‐inspired nonviral gene therapy that has promising clinical applications in melanoma treatment.

Intranasal Delivery of pGDNF DNA Nanoparticles Provides Neuroprotection in the Rat 6-Hydroxydopamine Model of Parkinson's Disease.

Glial cell line-derived neurotrophic factor (GDNF) gene therapy could offer a disease-modifying treatment for Parkinson's disease (PD). Here, we report that plasmid DNA nanoparticles (NPs) encoding human GDNF administered intranasally to rats induce transgene expression in the brain and protect dopamine neurons in a model of PD. To first test whether intranasal administration could transfect cells in the brain, rats were sacrificed 1week after intranasal pGDNF NPs or the naked plasmid. GDNF ELISA revealed significant increases in GDNF expression throughout the brain for both treatments. To assess whether expression was sufficient to protect dopamine neurons, naked pGDNF and pGDNF DNA NPs were given intranasally 1week before a unilateral 6-hydroxydopamine lesion in a rat model of PD. Three to four weeks after the lesion, amphetamine-induced rotational behavior was reduced, and dopaminergic fiber density and cell counts in the lesioned substantia nigra and nerve terminal density in the lesioned striatum were significantly preserved in rats given intranasal pGDNF. The NPs afforded a greater level of neuroprotection than the naked plasmid. These results provide proof-of-principle that intranasal administration of pGDNF DNA NPs can offer a non-invasive, non-viral gene therapy approach for early-stage PD.

Antitumor in situ vaccination effect of TNF\u03b1 and IL-12 plasmid DNA electrotransfer in a murine melanoma model

Gene electrotransfer (GET) is one of the most efficient non-viral gene therapy approaches for the localized transfer of multiple genes into tumors in vivo; therefore, it is especially promising for delivering different cytokines that are toxic if administered systemically. In this study, we used concomitant intratumoral GET of two cytokines: tumor necrosis factor alpha (TNFα), a potent cytotoxic cytokine to induce in situ vaccination, and interleukin 12 (IL-12), an immunostimulatory cytokine to boost the primed local immune response into a systemic one. After performing GET in murine melanoma tumors, both TNFα and IL-12 mRNA levels were significantly increased, which resulted in a pronounced delay in tumor growth of 27 days and a prolonged survival time of mice. An antitumor immune response was confirmed by extensive infiltration of immune cells in the tumor site, and expansion of the effector immune cells in the sentinel lymph nodes. Furthermore, the effect of in situ vaccination was indicated by the presence of vitiligo localized to the treatment area and resistance of the mice to secondary challenge with tumor cells. Intratumoral GET of two cytokines, one for in situ vaccination and one for an immune boost, proved feasible and effective in eliciting a potent and durable antitumor response; therefore, further studies of this approach are warranted.

Efficient Non-viral Gene Delivery into Human Hematopoietic Stem Cells by Minicircle Sleeping Beauty Transposon Vectors.

The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. We set out to improve SB gene transfer into hard-to-transfect human CD34+ cells by vectorizing the SB system components in the form of minicircles that are devoid of plasmid backbone sequences and are, therefore, significantly reduced in size. As compared to conventional plasmids, delivery of the SB transposon system as minicircle DNA is ∼20 times more efficient, and it is associated with up to a 50% reduction in cellular toxicity in human CD34+ cells. Moreover, providing the SB transposase in the form of synthetic mRNA enabled us to further increase the efficacy and biosafety of stable gene delivery into hematopoietic progenitors ex vivo. Genome-wide insertion site profiling revealed a close-to-random distribution of SB transposon integrants, which is characteristically different from gammaretroviral and lentiviral integrations in HSPCs. Transplantation of gene-marked CD34+ cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution, which was most efficient when the SB transposase was supplied as mRNA and nucleofected cells were maintained for 4–8 days in culture before transplantation. Collectively, implementation of minicircle and mRNA technologies allowed us to further refine the SB transposon system in the context of HSPC gene delivery to ultimately meet clinical demands of an efficient and safe non-viral gene therapy protocol.

Current Status of Nonviral Vectors for Gene Therapy in China.

With the growing interest in application of nonviral vectors for drug delivery, diagnosis, and imaging, progress has been made in the field of nonviral vector gene therapy in China. Nanobiotechnology studies are important to the development plan for China's research priorities in the 21st century; the National 973 Plan and "Strategic Priority Research Program" classify nanobiotechnology as a special project and give priority to supporting its development. From 2000 to 2017, many articles on nonviral vector gene therapy were published, and cancer gene therapy is one of the most active and important gene therapy research fields. Although the use of nonviral vectors for gene therapy faces enormous problems, Chinese scientists have also begun to realize both the challenges and opportunities that lie ahead. However, a nonviral gene therapy drug has yet to be approved for sale. Therefore, more work is needed in this field. The present review examines the progress and challenges of nonviral gene therapy in China. Efforts to optimize nonviral vectors with structural modifications and gene delivery systems are described, and clinical translation challenges are highlighted.

Optimizing Non-viral Gene Therapy Vectors for Delivery to Photoreceptors and Retinal Pigment Epithelial Cells.

Considerable progress has been made in the design and delivery of non-viral gene therapy vectors, but, like their viral counterparts, therapeutic levels of transgenes have not met the requirements for successful clinical applications so far. The biggest advantage of polymer-based nanoparticle vectors is the ease with which they can be modified to increase their ability to penetrate the cell membrane and target specific cells by simply changing the formulation of the nanoparticle compaction. We took advantage of this characteristic to improve transfection rates of our particles to meet the transgene levels which will be needed for future treatment of patients. For this study, we successfully investigated the possibility of our established pegylated polylysine particles to be administered via intravitreal rather than subretinal route to ease the damage during injection. We also demonstrated that our particles are flexible enough to sustain changes in the formulation to accommodate additional targeting sequences without losing their efficiency in transfecting neuronal cells in the retina. Together, these results give us the opportunity to even further improve our particles.

Delivery of modified mRNA encoding vesicular stomatitis virus matrix protein for colon cancer gene therapy.

Plasmid DNA based gene delivery has been widely utilized among both pre-clinical and clinical gene therapy studies. However, therapeutic efficiency is usually limited by the size and potential immune-stimulation issue of plasmid backbone. As an alternative form of genetic material, chemically modified messenger RNA (mRNA) provides a promising alternative to plasmid DNA. In this work, an in vitro transcription mRNA encoding vesicular stomatitis virus matrix protein (VSVMP) was delivered by a cationic liposome–protamine complex, resulting in high mRNA transporting and expression efficiency. The liposome–protamine complex delivered VSVMP mRNA strongly inhibits the growth of C26 tumor cells through inducing apoptosis, while obvious tumor regressions were achieved on both abdominal cavity metastatic and subcutaneous xenograft models in vivo with high safety. Our results also demonstrated that the liposome–protamine–mRNA complex was as potent as its plasmid DNA counterpart, showing strong potential in further colon cancer therapy.

Regression of prostate tumors after intravenous administration of lactoferrin-bearing polypropylenimine dendriplexes encoding TNF-α, TRAIL, and interleukin-12

The possibility of using gene therapy for the treatment of prostate cancer is limited by the lack of intravenously administered delivery systems able to safely and selectively deliver therapeutic genes to tumors. Given that lactoferrin (Lf) receptors are overexpressed on prostate cancer cells, we hypothesized that the conjugation of Lf to generation 3-diaminobutyric polypropylenimine dendrimer would improve its transfection and therapeutic efficacy in prostate cancer cells. In this study, we demonstrated that the intravenous administration of Lf-bearing DAB dendriplexes encoding TNFα resulted in the complete suppression of 70% of PC-3 and 50% of DU145 tumors over one month. Treatment with DAB-Lf dendriplex encoding TRAIL led to tumor suppression of 40% of PC-3 tumors and 20% of DU145 tumors. The treatment was well tolerated by the animals. Lf-bearing generation 3-polypropylenimine dendrimer is therefore a highly promising delivery system for non-viral gene therapy of prostate cancer.

Lipid coated chitosan-DNA nanoparticles for enhanced gene delivery

Chitosan as a polycationic non-viral vector for gene delivery has the advantage of being a biocompatible and biodegradable polymer. However, without laborious chemical modifications to its structure, it is of limited use as a gene delivery vehicle due to its low ability to efficiently transfect under physiological conditions. To address this problem, we developed novel liposome encapsulated chitosan nanoparticles; lipochitoplexes (LCPs). Chitosan nanoparticles (CsNPs) were obtained using the ionic gelation technique. For this purpose, an ultrapure low molecular weight chitosan with a high degree of deacetylation was cross-linked using polyanionic tripolyphosphate resulting in efficient entrapment of plasmid DNA (pDNA) inside the nanoparticles. LCPs were prepared by incubating chitosan nanoparticles together with anionic liposomes (DPPC/Cholesterol). The LCPs offered better pDNA protection, reduced cytotoxicity and at least twofold increase in the transfection efficiency under physiological conditions. The efficiency of our delivery vehicle was also proved in vivo in the chorioallantoic membrane model (CAM). LCPs were able to transfect the CAM without traumatising the surrounding blood vessels. This new biocompatible composite system devoid of chemical modifications, organic solvents and harsh production conditions makes it an optimal gene delivery vehicle for in vivo applications offering new insights into the field of non-viral gene therapy.

Effects of spinal non-viral interleukin-10 gene therapy formulated with d-mannose in neuropathic interleukin-10 deficient mice: Behavioral characterization, mRNA and protein analysis in pain relevant tissues

Studies show that spinal (intrathecal; i.t.) interleukin-10 (IL-10) gene therapy reverses neuropathic pain in animal models, and co-administration with the mannose receptor (MR; CD206) ligand d-mannose (DM) greatly improves therapeutic efficacy. However, the actions of endogenous IL-10 may be required for enduring pain control observed following i.t. IL-10 gene therapy, potentially narrowing the application of this non-viral transgene delivery approach. Here, we show that i.t. application of naked plasmid DNA expressing the IL-10 transgene co-injected with DM (DM/pDNA-IL-10) for the treatment of peripheral neuropathic pain in IL-10 deficient (IL-10 KO) mice results in a profound and prolonged bilateral pain suppression. Neuropathic pain is induced by unilateral sciatic chronic constriction injury (CCI), and while enduring relief of light touch sensitivity (mechanical allodynia) in both wild type (WT) and IL-10 KO mice was observed following DM/pDNA-IL-10 co-therapy, transient reversal from allodynia was observed following i.t. DM alone. In stably pain-relieved IL-10 KO mice given DM/pDNA-IL-10, mRNA for the IL-10 transgene is detected in the cauda equina and ipsilateral dorsal root ganglia (DRG), but not the lumbar spinal cord. Further, DM/pDNA-IL-10 application increases anti-inflammatory TGF-β1 and decreases pro-inflammatory TNF mRNA in the ipsilateral DRG compared to allodynic controls. Additionally, DM/pDNA-IL-10 treated mice exhibit decreased spinal pro-inflammatory mRNA expression for TNF, CCL2 (MCP-1), and for the microglial-specific marker TMEM119. Similarly, DM/pDNA-IL-10 treatment decreases immunoreactivity for the astrocyte activation marker GFAP in lumbar spinal cord dorsal horn. Despite transient reversal and early return to allodynia in DM-treated mice, lumbar spinal cord revealed elevated TNF, CCL2 and TMEM119 mRNA levels. Both MR (CD206) and IL-10 receptor mRNAs are increased in the DRG following CCI manipulation independent of injection treatment, suggesting that pathological conditions stimulate upregulation and availability of relevant receptors in critical anatomical regions required for the therapeutic actions of the DM/pDNA-IL-10 co-therapy. Taken together, the current report demonstrates that non-viral DM/pDNA-IL-10 gene therapy does not require endogenous IL-10 for enduring relief of peripheral neuropathic pain and does not require direct contact with the spinal cord dorsal horn for robust and enduring relief of neuropathic pain. Spinal non-viral DM/pDNA-IL-10 co-therapy may offer a framework for the development of non-viral gene therapeutic approaches for other diseases of the central nervous system.

222 Formulation of a novel high performance non-viral gene therapy for recessive dystrophic epidermolysis bullosa

222 Formulation of a novel high performance non-viral gene therapy for recessive dystrophic epidermolysis bullosa

ECM turnover-stimulated gene delivery through collagen-mimetic peptide-plasmid integration in collagen.

In this work, we demonstrate a novel approach for stably integrating DNA into collagen scaffolds to exploit the natural process of collagen remodelling for high efficiency non-viral gene delivery. The incorporation of CMPs into DNA polyplexes, coupled with the innate affinity between CMPs and collagen, not only permitted improved control over polyplex retention and release, but also provided a series of substantial and highly unique benefits via the stable and persistent linkage between CMP-polyplexes and collagen fragments. Specifically, CMP-modification of polyplexes was demonstrated to (i) control release for nearly a month, (ii) improve vector stability under physiological-like conditions, and (iii) provide ligands able to efficiently transfer genes via endocytic collagen pathways. These unique properties overcome key barriers inhibiting non-viral gene therapy.

Multiplexed RNAi therapy against brain tumor-initiating cells via lipopolymeric nanoparticle infusion delays glioblastoma progression

Brain tumor-initiating cells (BTICs) have been identified as key contributors to therapy resistance, recurrence, and progression of diffuse gliomas, particularly glioblastoma (GBM). BTICs are elusive therapeutic targets that reside across the blood-brain barrier, underscoring the urgent need to develop novel therapeutic strategies. Additionally, intratumoral heterogeneity and adaptations to therapeutic pressure by BTICs impede the discovery of effective anti-BTIC therapies and limit the efficacy of individual gene targeting. Recent discoveries in the genetic and epigenetic determinants of BTIC tumorigenesis offer novel opportunities for RNAi-mediated targeting of BTICs. Here we show that BTIC growth arrest in vitro and in vivo is accomplished via concurrent siRNA knockdown of four transcription factors (SOX2, OLIG2, SALL2, and POU3F2) that drive the proneural BTIC phenotype delivered by multiplexed siRNA encapsulation in the lipopolymeric nanoparticle 7C1. Importantly, we demonstrate that 7C1 nano-encapsulation of multiplexed RNAi is a viable BTIC-targeting strategy when delivered directly in vivo in an established mouse brain tumor. Therapeutic potential was most evident via a convection-enhanced delivery method, which shows significant extension of median survival in two patient-derived BTIC xenograft mouse models of GBM. Our study suggests that there is potential advantage in multiplexed targeting strategies for BTICs and establishes a flexible nonviral gene therapy platform with the capacity to channel multiplexed RNAi schemes to address the challenges posed by tumor heterogeneity.

Nonviral cancer gene therapy: Delivery cascade and vector nanoproperty integration.

Gene therapy represents a promising cancer treatment featuring high efficacy and limited side effects, but it is stymied by a lack of safe and efficient gene-delivery vectors. Cationic polymers and lipid-based nonviral gene vectors have many advantages and have been extensively explored for cancer gene delivery, but their low gene-expression efficiencies relative to viral vectors limit their clinical translations. Great efforts have thus been devoted to developing new carrier materials and fabricating functional vectors aimed at improving gene expression, but the overall efficiencies are still more or less at the same level. This review analyzes the cancer gene-delivery cascade and the barriers, the needed nanoproperties and the current strategies for overcoming these barriers, and outlines PEGylation, surface-charge, size, and stability dilemmas in vector nanoproperties to efficiently accomplish the cancer gene-delivery cascade. Stability, surface, and size transitions (3S Transitions) are proposed to resolve those dilemmas and strategies to realize these transitions are comprehensively summarized. The review concludes with a discussion of the future research directions to design high-performance nonviral gene vectors.