Gene Delivery Process Research Articles

Macrocycle-Based Supramolecular Drug Delivery Systems: A Concise Review.

Efficient delivery of therapeutic agents to the lesion site or specific cells is an important way to achieve "toxicity reduction and efficacy enhancement". Macrocycles have always provided many novel ideas for drug or gene loading and delivery processes. Specifically, macrocycles represented by crown ethers, cyclodextrins, cucurbit[n]urils, calix[n]arenes, and pillar[n]arenes have unique properties, which are different cavity structures, good biocompatibility, and good stability. Benefited from these diverse properties, a variety of supramolecular drug delivery systems can be designed and constructed to effectively improve the physical and chemical properties of guest molecules as needed. This review provides an outlook on the current application status and main limitations of macrocycles in supramolecular drug delivery systems.

Creation of biomimetic polyethylenimine quantum dots nanocomposites for traceable and targeted DNA delivery

The potential of gene therapy for curing cancer is promising, but the lack of safe and effective gene delivery systems limits its clinical application. Polyethyleneimine (PEI) is a non-viral gene delivery vector with potential, but it has high toxicity and lacks targeting capabilities. To address these challenges, this study developed organic quantum dots (PQDs) based on different molecular weight PEI using a hydrothermal method. The PQDs showed significant quantum dot (QDs) fluorescence characteristics, lower cytotoxicity, and higher transfection efficiency compared to PEI of the same molecular weight. Among the PQDs, P25QDs (made from PEI25k) demonstrated the highest gene transfection efficiency. Furthermore, encapsulating P25QDs/DNA using different natural cell membranes resulted in biomimetic nanocomposites CM-PQDs/DNA that maintained excellent QDs fluorescence characteristics, lower cytotoxicity, and higher cell uptake efficiency compared to PEI25k/DNA and P25QDs/DNA complexes. Interestingly, these cell membrane-encapsulated nanocomposites also showed significant homologous targeting, with HeLa-PQDs/DNA and RAW-PQDs/DNA exhibiting higher cellular uptake and transfection efficiency in HeLa cells and 4T1 cells, respectively. These findings suggest that converting PEI into quantum dots can produce gene carriers with improved safety and transfection efficiency, as well as enable fluorescence tracing of gene delivery processes, which is significant for advancing the use of PEI as a gene vector.

Synthesis and evaluation of gene delivery vectors based on PEI-modified metal-organic framework (MOF) nanoparticles.

Zirconium-based metal-organic frameworks (MOFs) nanostructures, due to their capability of easy surface modification, are considered interesting structures for delivery. In the present study, the surfaces of UIO-66 and NH2-UIO-66 MOFs were modified by polyethyleneimine (PEI) 10000 Da, and their efficiency for plasmid delivery was evaluated. Two different approaches, were employed to prepare surface-modified nanoparticles. The physicochemical characteristics of the resulting nanoparticles, as well as their transfection efficiency and cytotoxicity, were investigated on the A549 cell line. The sizes of DNA/nanocarriers for PEI-modified UIO-66 (PEI-UIO-66) were between 212-291 nm and 267-321 nm for PEI 6-bromohexanoic acid linked UIO-66 (PEI-HEX-UIO-66). The zeta potential of all was positive with the ranges of +16 to +20 mV and +23 to +26 mV for PEI-UIO-66 and PEI-HEX-UIO-66, respectively. Cellular assay results showed that the PEI linking method had a higher rate of gene transfection efficiency with minimal cytotoxicity than the wet impregnation method. The difference between transfection of modified nanoparticles compared to the PEI 10 kDa was not significant but the PEI-HEX-UIO-66 showed less cytotoxicity. The present study suggested that the post-synthetic modification of MOFs with PEI 10000 Da through EDC/NHS+6-bromohexanoic acid reaction can be considered as an effective approach for modifying MOFs' structure in order to obtain nanoparticles with better biological function in the gene delivery process.

Growth factors and growth factor gene therapies for treating chronic wounds

AbstractChronic wounds are an unmet clinical need affecting millions of patients globally, and current standards of care fail to consistently promote complete wound closure and prevent recurrence. Disruptions in growth factor signaling, a hallmark of chronic wounds, have led researchers to pursue growth factor therapies as potential supplements to standards of care. Initial studies delivering growth factors in protein form showed promise, with a few formulations reaching clinical trials and one obtaining clinical approval. However, protein‐form growth factors are limited by instability and off‐target effects. Gene therapy offers an alternative approach to deliver growth factors to the chronic wound environment, but safety concerns surrounding gene therapy as well as efficacy challenges in the gene delivery process have prevented clinical translation. Current growth factor delivery and gene therapy approaches have primarily used single growth factor formulations, but recent efforts have aimed to develop multi‐growth factor approaches that are better suited to address growth factor insufficiencies in the chronic wound environment, and these strategies have demonstrated improved efficacy in preclinical studies. This review provides an overview of chronic wound healing, emphasizing the need and potential for growth factor therapies. It includes a summary of current standards of care, recent advances in growth factor, cell‐based, and gene therapy approaches, and future perspectives for multi‐growth factor therapeutics.

Enhanced gene transfection efficacy and safety through granular hydrogel mediated gene delivery process

Enhanced gene transfection efficacy and safety through granular hydrogel mediated gene delivery process

Advances and Challenges of Stimuli-Responsive Nucleic Acids Delivery System in Gene Therapy.

Gene therapy has emerged as a powerful tool to treat various diseases, such as cardiovascular diseases, neurological diseases, ocular diseases and cancer diseases. In 2018, the FDA approved Patisiran (the siRNA therapeutic) for treating amyloidosis. Compared with traditional drugs, gene therapy can directly correct the disease-related genes at the genetic level, which guarantees a sustained effect. However, nucleic acids are unstable in circulation and have short half-lives. They cannot pass through biological membranes due to their high molecular weight and massive negative charges. To facilitate the delivery of nucleic acids, it is crucial to develop a suitable delivery strategy. The rapid development of delivery systems has brought light to the gene delivery field, which can overcome multiple extracellular and intracellular barriers that prevent the efficient delivery of nucleic acids. Moreover, the emergence of stimuli-responsive delivery systems has made it possible to control the release of nucleic acids in an intelligent manner and to precisely guide the therapeutic nucleic acids to the target site. Considering the unique properties of stimuli-responsive delivery systems, various stimuli-responsive nanocarriers have been developed. For example, taking advantage of the physiological variations of a tumor (pH, redox and enzymes), various biostimuli- or endogenous stimuli-responsive delivery systems have been fabricated to control the gene delivery processes in an intelligent manner. In addition, other external stimuli, such as light, magnetic fields and ultrasound, have also been employed to construct stimuli-responsive nanocarriers. Nevertheless, most stimuli-responsive delivery systems are in the preclinical stage, and some critical issues remain to be solved for advancing the clinical translation of these nanocarriers, such as the unsatisfactory transfection efficiency, safety issues, complexity of manufacturing and off-target effects. The purpose of this review is to elaborate the principles of stimuli-responsive nanocarriers and to emphasize the most influential advances of stimuli-responsive gene delivery systems. Current challenges of their clinical translation and corresponding solutions will also be highlighted, which will accelerate the translation of stimuli-responsive nanocarriers and advance the development of gene therapy.

Nanotechnology-enabled gene delivery for cancer and other genetic diseases

Introduction Despite gene therapy is ideal for genetic abnormality-related diseases, the easy degradation, poor targeting, and inefficiency in entering targeted cells are plaguing the effective delivery of gene therapy. Viral and non-viral vectors have been used for delivering gene therapeutics in vivo by safeguarding nucleic acid agents to target cells and to reach the specific intracellular location. A variety of nanotechnology-enabled safe and efficient systems have been successfully developed to improve the targeting ability for effective therapeutic delivery of genetic drugs. Areas covered In this review, we outline the multiple biological barriers associated with gene delivery process, and highlight recent advances to gene therapy strategy in vivo, including gene correction, gene silencing, gene activation and genome editing. We point out current developments and challenges exist of non-viral and viral vector systems in association with chemical and physical gene delivery technologies and their potential for the future. Expert opinion This review focuses on the opportunities and challenges to various gene therapy strategy, with specific emphasis on overcoming the challenges through the development of biocompatibility and smart gene vectors for potential clinical application.

Hyper-Branched Cationic Cyclodextrin Polymers for Improving Plasmid Transfection in 2D and 3D Spheroid Cells.

In this article, we used monolayer two dimensional (2D) and 3D multicellular spheroid models to improve our understanding of the gene delivery process of a new modified cationic hyper-branched cyclodextrin-based polymer (Ppoly)-loaded plasmid encoding Enhanced Green Fluorescent Protein (EGFP). A comparison between the cytotoxicity effect and transfection efficiency of the plasmid DNA (pDNA)-loaded Ppoly system in 2D and 3D spheroid cells determined that the transfection efficiency and cytotoxicity of Ppoly-pDNA nanocomplexes were lower in 3D spheroids than in 2D monolayer cells. Furthermore, histopathology visualization of Ppoly-pDNA complex cellular uptake in 3D spheroids demonstrated that Ppoly penetrated into the inner layers. This study indicated that the Ppoly, as a non-viral gene delivery system in complex with pDNA, is hemocompatible, non-toxic, high in encapsulation efficiency, and has good transfection efficiency in both 2D and 3D cell cultures compared to free pDNA and lipofectamine (as the control).

Biocompatible Iron Oxide Nanoparticles for Targeted Cancer Gene Therapy: A Review.

In recent years, gene therapy has made remarkable achievements in tumor treatment. In a successfully cancer gene therapy, a smart gene delivery system is necessary for both protecting the therapeutic genes in circulation and enabling high gene expression in tumor sites. Magnetic iron oxide nanoparticles (IONPs) have demonstrated their bright promise for highly efficient gene delivery target to tumor tissues, partly due to their good biocompatibility, magnetic responsiveness, and extensive functional surface modification. In this review, the latest progress in targeting cancer gene therapy is introduced, and the unique properties of IONPs contributing to the efficient delivery of therapeutic genes are summarized with detailed examples. Furthermore, the diagnosis potentials and synergistic tumor treatment capacity of IONPs are highlighted. In addition, aiming at potential risks during the gene delivery process, several strategies to improve the efficiency or reduce the potential risks of using IONPs for cancer gene therapy are introduced and addressed. The strategies and applications summarized in this review provide a general understanding for the potential applications of IONPs in cancer gene therapy.

Red fluorescent AIEgens based multifunctional nonviral gene vectors for the efficient combination of gene therapy and photodynamic therapy in anti-cancer

Precise molecular engineering of AIEgens-based cationic delivery systems for high transfection efficiency (TE) and effective photodynamic therapy (PDT) holds a huge potential for cancer treatment. Herein, three amphiphiles (DT-C6/8/12-M) consisting of di(triazole-[12]aneN3) (M) and 1,1-dicyano-2-phenyl-2-(4-diphenylamino)phenyl-ethylene (DT) units have been developed to achieve luminescent tracking, efficient TE, and effective PDT in vitro and in vivo. These compounds exhibited strong aggregated induced emission (AIE) at 630 nm and mega Stokes shifts of up to 160 nm. They were able to bind DNA into nanoparticles with suitable sizes, positive surface potential, and good biocompatibility in the presence of DOPE. Among them, vector DT-C12-M/DOPE with n-dodecyl linker achieved a transfection efficiency as high as 42.3 folds that of Lipo2000 in PC-3 cell lines. DT-C12-M/DOPE exhibited the capability of successful endo/lysosomal escape and rapid nuclear delivery of pDNA, and the gene delivery process was clearly monitored via confocal laser scanning microscopy. Moreover, efficient reactive oxygen species (ROS) generation by DT-C12-M upon light irradiation led to effective PDT in vitro . We further show that combination of p53 gene therapy and PDT dramatically enhanced cancer therapeutic outcome in vivo. This “three birds, one stone” strategy offers a novel and promising approach for real-time tracking of gene delivery and better cancer treatment

Multifunctional amphiphilic peptide dendrimer as nonviral gene vectors for effective cancer therapy via combined gene/photodynamic therapies

Gene therapy holds great promise for treatment of gene-associated diseases. However, safe and successful clinical application urgently requires further advancement of constructing efficient delivery systems. Herein, three amphiphilic peptide dendrimers (TTC-L-KRR/KKK/KHH), containing the natural amino acid residues (lysine K, arginine R, and histidine H) and AIE-based photosensitizer (tetraphenylethenethiophene modified cyanoacrylate, TTC) modified with alkyl chain (L), have been designed and prepared for improving therapeutic potency via the combination of gene therapy (GT) and photodynamic therapy (PDT). All three compounds possessed typical aggregation-induced emission (AIE) characteristics and ultralow critical micelle concentrations (CMCs). The liposomes consisting of amphiphilic peptide dendrimers and dioleoylphosphatidylethanolamine (DOPE) can effectively bind DNA into nanoparticles with appropriate sizes, regular morphology and good biocompatibility. Among them, liposomes TTC-L-KKK/DOPE exhibited the highest transfection efficiency up to 5.7-fold as compared with Lipo2000 in HeLa cells. Meanwhile, rapid endocytosis, successful endo/lysosomal escape, gene release and rapid nuclear delivery of DNA revealed the superiority of liposomes TTC-L-KKK/DOPE during gene delivery process. More importantly, efficient reactive oxygen species (ROS) generation by TTC-L-KKK/DOPE led to effective PDT, thus improving therapeutic potency via combining with p53 mediated-gene therapy. Our work brought novel insight and direction for the construction of bio-safe and bio-imaging liposome as the multifunctional nonviral gene vectors for the effective combined gene/photodynamic therapies.

Abstract 2821: Incorporation of intrinsic checkpoint blockade enhances functionality of multigenic autologous UltraCAR-T® cells manufactured using non-viral gene delivery and rapid manufacturing process

Abstract Current chimeric antigen receptor (CAR)-T manufacturing utilizes viral vectors and extensive ex vivo expansion at large central facilities leading to an exhausted CAR-T phenotype, high costs and long vein-to-vein times. While allogeneic CAR-T can reduce delays in patient treatment, they require extensive manipulation of donor cells, severe lymphodepletion and demonstrate short persistence limiting their therapeutic window. The UltraCAR-T® platform is designed to overcome these limitations by utilizing non-viral multigene delivery and a rapid, decentralized manufacturing process without ex vivo activation or expansion of T cells. Patient’s own T cells are collected and manufactured at the medical center and re-infused one day after gene transfer. Here we describe the next generation UltraCAR-T platform that addresses the inhibitory tumor microenvironment by incorporating a novel mechanism for intrinsic downregulation of one or more checkpoint inhibitor (CPI) genes. Our design achieves intrinsic CPI blockade without gene editing and is aimed at avoiding systemic toxicity and the high cost of combining CPI antibodies. Next generation UltraCAR-T cells simultaneously express CAR, membrane bound IL-15 (mbIL15), kill switch, and incorporate intrinsic CPI blockade. To illustrate the ability of this platform, we designed exemplary non-viral transposons to generate UltraCAR-T cells against multiple tumor targets incorporating intrinsic blockade of either one (PD-1) or two (PD-1 and TIGIT) CPI genes. Healthy donor T cells were transfected using the UltraPorator™ electroporation system to manufacture UltraCAR-T cells without ex vivo activation or expansion. The co-expression of CAR, mbIL15 and kill switch was confirmed by flow cytometry and western blot. Activated UltraCAR-T showed significant reduction in CPI gene expression compared to control CAR-T cells lacking the CPI blockade and did not show unintended off-target activity. Downregulation of CPI gene(s) on UltraCAR-T enhanced cytotoxicity and inflammatory cytokine production, especially at low effector to target (E:T) cell ratios, when co-cultured with PD-L1+/CD155+ tumor cells. Single-cell cytokine proteomics showed significant increase in polyfunctionality of UltraCAR-T with intrinsic downregulation of CPI gene(s). In vivo, a single infusion of receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific UltraCAR-T with intrinsic PD-1 blockade resulted in rapid expansion, an increase in preferred T cell memory (TSCM/TCM) populations, and significantly improved overall survival of ROR1+ PD-L1+ tumor bearing mice. These preclinical data highlight the improved efficacy of incorporating intrinsic CPI blockade in UltraCAR-T cells using non-viral gene delivery and an established rapid, decentralized manufacturing process. Citation Format: Tim Chan, Cheryl Bolinger, Sean Scott, Mengyan Du, Carol Poortman, Byron Koenitzer, Taranjit Athwal, Lindsey Shepard, R. Daniel Slone, Shourik Dutta, Steven Zilko, James M. Dunleavey, Giorgio Zenere, Jacques Plummer, Bernward Klocke, Christian Zinser, Shamim Ahmad, Douglas E. Brough, Rutul R. Shah, Helen Sabzevari. Incorporation of intrinsic checkpoint blockade enhances functionality of multigenic autologous UltraCAR-T® cells manufactured using non-viral gene delivery and rapid manufacturing process [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2821.

Modeling the gene delivery process of the needle array-based tissue nanotransfection.

Hollow needle array-based tissue nanotransfection (TNT) presents an in vivo transfection approach that directly translocate exogeneous genes to target tissues by using electric pulses. In this work, the gene delivery process of TNT was simulated and experimentally validated. We adopted the asymptotic method and cell-array-based model to investigate the electroporation behaviors of cells within the skin structure. The distribution of nonuniform electric field across the skin results in various electroporation behavior for each cell. Cells underneath the hollow microchannels of the needle exhibited the highest total pore numbers compared to others due to the stronger localized electric field. The percentage of electroporated cells within the skin structure, with pore radius over 10 nm, increases from 25% to 82% as the applied voltage increases from 100 to 150 V/mm. Furthermore, the gene delivery behavior across the skin tissue was investigated through the multilayer-stack-based model. The delivery distance increased nonlinearly as the applied voltage and pulse number increased, which mainly depends on the diffusion characteristics and electric conductivity of each layer. It was also found that the skin is required to be exfoliated prior to the TNT procedure to enhance the delivery depth. This work provides the foundation for transition from the study of murine skin to translation use in large animals and human settings.

Supramolecular Surface Functionalization of Iron Oxide Nanoparticles with α-Cyclodextrin-Based Cationic Star Polymer for Magnetically-Enhanced Gene Delivery.

Supramolecular polymers formed through host–guest complexation have inspired many interesting developments of functional materials for biological and biomedical applications. Here, we report a novel design of a non-viral gene delivery system composed of a cationic star polymer forming supramolecular complexes with the surface oleyl groups of superparamagnetic iron oxide nanoparticles (SPIONs), for magnetically enhanced delivery of DNA into mammalian cells. The cationic star polymer was synthesized by grafting multiple oligoethylenimine (OEI) chains onto an α-cyclodextrin (α-CD) core. The SPIONs were synthesized from iron(III) acetylacetonate and stabilized by hydrophobic oleic acid and oleylamine in hexane, which were characterized in terms of their size, structure, morphology, and magnetic properties. The synthesized magnetic particles were found to be superparamagnetic, making them a suitable ferrofluid for biological applications. In order to change the hydrophobic surface of the SPIONs to a hydrophilic surface with functionalities for plasmid DNA (pDNA) binding and gene delivery, a non-traditional but simple supramolecular surface modification process was used. The α-CD-OEI cationic star polymer was dissolved in water and then mixed with the SPIONs stabilized in hexane. The SPIONs were “pulled” into the water phase through the formation of supramolecular host–guest inclusion complexes between the α-CD unit and the oleyl surface of the SPIONs, while the surface of the SPIONs was changed to OEI cationic polymers. The α-CD-OEI-SPION complex could effectively bind and condense pDNA to form α-CD-OEI-SPION/pDNA polyplex nanoparticles at the size of ca. 200 nm suitable for delivery of genes into cells through endocytosis. The cytotoxicity of the α-CD-OEI-SPION complex was also found to be lower than high-molecular-weight polyethylenimine, which was widely studied previously as a standard non-viral gene vector. When gene transfection was carried out in the presence of an external magnetic field, the α-CD-OEI-SPION/pDNA polyplex nanoparticles greatly increased the gene transfection efficiency by nearly tenfold. Therefore, the study has demonstrated a facile two-in-one method to make the SPIONs water-soluble as well as functionalized for enhanced magnetofection.

Mechanical Stimulation after Centrifuge‐Free Nano‐Electroporative Transfection Is Efficient and Maintains Long‐Term T Cell Functionalities (Small 38/2021)

Transfection In article number 2103198, Andy Tay and Nicholas Melosh illustrate the use of high aspect-ratio hollow nanostructures coupled with transient electroporation for electrophoretic delivery of DNA into T cell. Magnetic stimulation was added subsequently to enhance intracellular DNA transport towards the nucleus to achieve high transfection efficiency. Facilitated by magnetic manipulation, the whole gene delivery process is centrifuge-free, making it compatible with end-to-end cell manufacturing.

DNA Condensation Triggered by the Synergistic Self-Assembly of Tetraphenylethylene-Viologen Aggregates and CT-DNA.

Development of small organic chromophores as DNA condensing agents, which explore supramolecular interactions and absorbance or fluorescence-based tracking of condensation and gene delivery processes, is in the initial stages. Herein, we report the synthesis and electrostatic/groove binding interaction–directed synergistic self-assembly of the aggregates of two viologen-functionalized tetraphenylethylene (TPE-V) molecules with CT-DNA and subsequent concentration-dependent DNA condensation process. TPE-V molecules differ in their chemical structure according to the number of viologen units. Photophysical and morphological studies have revealed the interaction of the aggregates of TPE-V in Tris buffer with CT-DNA, which transforms the fibrous network structure of CT-DNA to partially condensed beads-on-a-string-like arrangement with TPE-V aggregates as beads via electrostatic and groove binding interactions. Upon further increasing the concentration of TPE-V, the “beads-on-a-string”-type assembly of TPE-V/CT-DNA complex changes to completely condensed compact structures with 40–50 nm in diameter through the effective charge neutralization process. Enhancement in the melting temperature of CT-DNA, quenching of the fluorescence emission of ethidium bromide/CT-DNA complex, and the formation of induced CD signal in the presence of TPE-V molecules support the observed morphological changes and thereby verify the DNA condensation abilities of TPE-V molecules. Decrease in the hydrodynamic size, increase in the zeta potential value with the addition of TPE-V molecules to CT-DNA, failure of TPE-V/cucurbit(8)uril complex to condense CT-DNA, and the enhanced DNA condensation ability of TPE-V2 with two viologen units compared to TPE-V1 with a single viologen unit confirm the importance of positively charged viologen units in the DNA condensation process. Initial cytotoxicity analysis on A549 cancer and WI-38 normal cells revealed that these DNA condensing agents are non-toxic in nature and hence could be utilized in further cellular delivery studies.

Interaction kinetics of peptide lipids-mediated gene delivery

BackgroundDuring the course of gene transfection, the interaction kinetics between liposomes and DNA is speculated to play very important role for blood stability, cellular uptake, DNA release and finally transfection efficiency.ResultsAs cationic peptide liposomes exhibited great gene transfer activities both in vitro and in vivo, two peptide lipids, containing a tri-ornithine head (LOrn3) and a mono-ornithine head (LOrn1), were chosen to further clarify the process of liposome-mediated gene delivery in this study. The results show that the electrostatically-driven binding between DNA and liposomes reached nearly 100% at equilibrium, and high affinity of LOrn3 to DNA led to fast binding rate between them. The binding process between LOrn3 and DNA conformed to the kinetics equation: y = 1.663631 × exp (− 0.003427x) + 6.278163. Compared to liposome LOrn1, the liposome LOrn3/DNA lipoplex exhibited a faster and more uniform uptake in HeLa cells, as LOrn3 with a tri-ornithine peptide headgroup had a stronger interaction with the negatively charged cell membrane than LOrn1. The efficient endosomal escape of DNA from LOrn3 lipoplex was facilitated by the acidity in late endosomes, resulting in broken carbamate bonds, as well as the “proton sponge effect” of the lipid.ConclusionsThe interaction kinetics is a key factor for DNA transfection efficiency. This work provided insights into peptide lipid-mediated DNA delivery that could guide the development of the next generation of delivery systems for gene therapeutics.

Unprecedented Self-Assembly in Dilute Aqueous Solution of Polyethyleneimine: Formation of Fibrillar Network

Polyethyleneimine (PEI), a cationic polyelectrolyte, finds great utility as a nonviral gene transfection vector. The mechanism of gene delivery process across the cell membrane through PEI-based po...

Enhancers in polymeric nonviral gene delivery systems

AbstractGene therapy is a promising therapeutic tool for cancers and inherited diseases. For successful gene therapy, gene delivery systems must be designed reasonably to allow DNA or other nuclear acids to be taken up by the cancer cells and then transported to target location for function. In the research of gene delivery systems, polymeric nonviral carriers for genes have attracted much attention. However, polymeric gene delivery systems suffer from low transfection efficiency. Various strategies have been developed for improving efficacy of polymeric gene delivery systems. These strategies can be categorized into covalent strategies and noncovalent strategies according to the way how the functional unit is combined with the carrier. Noncovalent strategies in improving gene transfection efficiency are simply mixing additional functional components with carriers. These relatively independent functional materials are named gene delivery enhancers here. In this review, we focus on enhancers in polymeric gene delivery systems. We discuss structures and enhancement mechanisms of enhancers in the order of the stages in which they function in gene delivery process. Barriers of in vitro gene delivery are also reviewed briefly.

Abstract 6593: PRGN-3005 UltraCAR-T™: multigenic CAR-T cells generated using non-viral gene delivery and rapid manufacturing process for the treatment of ovarian cancer

Abstract Ovarian cancer is the most lethal of gynecologic cancers. Chimeric antigen receptor (CAR)-T cells have improved clinical outcomes in hematological malignancies; however similar responses have not yet been achieved for solid tumors. Furthermore, traditional methods for CAR-T manufacturing utilize viral vectors and weeks of ex vivo expansion to achieve clinically relevant cell numbers. This leads to CAR-T cells with an exhausted T cell phenotype, high manufacturing costs, and treatment delays. We have developed the UltraCAR-T™ platform to overcome these limitations by utilizing an advanced non-viral gene delivery system and a rapid, decentralized manufacturing process. Our non-viral gene delivery platform is based on Sleeping Beauty transposon system precision engineered for optimal delivery of multiple genes of UltraCAR-T. UltraCAR-T cells are manufactured at medical center's cGMP facility using autologous T cells and administered back to patient only one day after gene transfer. PRGN-3005 UltraCAR-T cells simultaneously co-express (i) a CAR specifically targeting MUC16 which is highly expressed on ovarian tumors with limited normal tissue expression; (ii) membrane bound IL-15 (mbIL15) for enhanced in vivo expansion and persistence; and (iii) a kill switch for improved therapeutic control. Healthy donor T cells were transfected with non-viral system encoding CAR, mbIL15 and kill switch to generate PRGN-3005 cells. Expression of transgenes was confirmed by flow cytometry and western blot analyses. PRGN-3005 demonstrated a robust MUC16-specific cytotoxicity of ovarian cancer cells in vitro. PRGN-3005 exhibited a stem-cell like memory phenotype, significant improvement in survival even in absence of exogenous cytokines and lacked autonomous proliferation in vitro in the absence of MUC16 compared to conventional CAR-T cells, demonstrating beneficial effects of mbIL15 on phenotype and persistence of UltraCAR-T cells. Furthermore, we demonstrated that PRGN-3005 cells were selectively and effectively eliminated by kill switch activator antibody via ADCC. For in vivo anti-tumor efficacy of PRGN-3005, an ovarian cancer model of MUC16+ SK-OV-3 cell line was evaluated in NSG mice. In tumor-bearing mice, a single administration of PRGN-3005 one day after gene transfer resulted rapid in vivo expansion and significant increase in UltraCAR-T cells, in particular T stem cell memory (TSCM) population compared to mbIL15neg CAR-T cells. PRGN-3005 demonstrated significantly higher anti-tumor effect (p<0.01) compared to mbIL15neg CAR-T and led to complete tumor regression in all treated mice. PRGN-3005 UltraCAR-T administered one day after non-viral gene transfer has superior anti-tumor efficacy and persistence compared traditional CAR-T cells and represent a promising opportunity for ovarian cancer treatment. Based on these results, FDA has approved an IND application and the first in human PRGN-3005 Phase I clinical trial for advanced ovarian cancer is currently under evaluation (NCT03907527). Citation Format: Tim Chan, Marion Chakiath, Lindsey Shepard, Simon Metenou, Fernando Carvajal-Borda, Jose Velez, Adeline Govekung, Jacques Plummer, Carol Poortman, Padma Kamineny, Xiaohong Ma, Rutul R. Shah, Mary L. Disis, Helen Sabzevari. PRGN-3005 UltraCAR-T™: multigenic CAR-T cells generated using non-viral gene delivery and rapid manufacturing process for the treatment of ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6593.