Gene Delivery Methods Research Articles

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection.

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

Atrial Gene Painting in Large Animal Model of Atrial Fibrillation.

Gene therapy appears promising as a targeted treatment of cardiac diseases. Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and also a major contributor to stroke, heart failure, and death. Mechanisms that initiate and sustain AF are associated with structural and electrophysiological remodeling in the whole atria. Selection of the appropriate gene delivery method is critical for transduction efficacy. The ideal gene delivery method to manage AF should provide widespread and sufficient exposure to the transgene in atria only that safely maintains the homeostasis of the heart without off-target expression. All these requirements can be achieved using atrial gene painting that is directly applied to the atrial epicardial surface. In this chapter, we present the advantages of atrial gene painting and the experimental method, as applied to a large animal model of AF.

Labeling Endogenous Proteins Using CRISPR-mediated Insertion of Exon (CRISPIE).

The CRISPR/Cas9 technology has transformed our ability to edit eukaryotic genomes. Despite this breakthrough, it remains challenging to precisely knock-in large DNA sequences, such as those encoding a fluorescent protein, for labeling or modifying a target protein in post-mitotic cells. Previous efforts focusing on sequence insertion to the protein coding sequence often suffer from insertions/deletions (INDELs) resulting from the efficient non-homologous end joining pathway (NHEJ). To overcome this limitation, we have developed CRISPR-mediated insertion of exon (CRISPIE). CRISPIE circumvents INDELs and other editing errors by inserting a designer exon flanked by adjacent intron sequences into an appropriate intronic location of the targeted gene. Because INDELs at the insertion junction can be spliced out, "CRISPIEd" genes produce precisely edited mRNA transcripts that are virtually error-free. In part due to the elimination of INDELs, high-efficiency labeling can be achieved in vivo. CRISPIE is compatible with both N- and C-terminal labels, and with all common transfection methods. Importantly, CRISPIE allows for later removal of the protein modification by including exogenous single-guide RNA (sgRNA) sites in the intronic region of the donor module. This protocol provides the detailed CRISPIE methodology, using endogenous labeling of β-actin in human U-2 OS cells with enhanced green fluorescent protein (EGFP) as an example. When combined with the appropriate gene delivery methods, the same methodology can be applied to label post-mitotic neurons in culture and in vivo. This methodology can also be readily adapted for use in other gene editing contexts.

Surgical Methods for Cardiac Gene Delivery in Large Animals.

This chapter describes main strategies of surgical gene delivery in large animals. Existing methods of cardiac gene transfer can be classified by the site of injection, interventional approach, and type of cardiac circulation at the time of transfer. Randomized clinical trials have suggested that the therapeutic benefits of gene therapy are not as substantial as expected from animal studies. This discordance in results is largely due to gene delivery methods that may be effective in small animals but are not scalable to larger species and, therefore, cannot transduce a sufficient fraction of myocytes to establish long-term clinical efficacy. Ideally, an optimized gene transfer should incorporate the following: a closed-loop recirculation for extended transgene residence time; vector washout form the vascular system after transfer to prevent collateral expression; use of methods to increase myocardial transcapillary gradient for viral particles for a better transduction, probably retrograde route of gene delivery through the coronary venous system; and myocardial ischemic preconditioning.

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.

Optimization of Synthesis of Modified mRNA.

Modified mRNA (modRNA) is a safe and effective vector for gene-based therapies. Notably, the safety of modRNA has been validated through COVID-19 vaccines which incorporate modRNA technology to translate spike proteins. Alternative gene delivery methods using plasmids, lentiviruses, adenoviruses, and adeno-associated viruses have suffered from key challenges such as genome integration, delayed and uncontrolled expression, and immunogenic responses. However, modRNA poses no risk of genome integration, has transient and rapid expression, and lacks an immunogenic response. Our lab utilizes modRNA-based therapies to promote cardiac regeneration following myocardial infarction and heart failure. We have also developed and refined an optimized and economical method for synthesis of modRNA. Here, we provide an updated methodology with improved translational efficiency for in vitro and in vivo application.

Cardiac Gene Delivery in Large Animal Models: Antegrade Techniques.

Percutaneous antegrade coronary injection is among the least invasive cardiac selective gene delivery methods. However, the transduction efficiency of a simple bolus antegrade injection is quite low. In order to improve transduction efficiency in antegrade intracoronary delivery, several additional approaches have been proposed.In this chapter, we will describe the important elements associated with intracoronary delivery methods and present protocols for three different catheter-based antegrade gene delivery techniques in a preclinical large animal model. This is the second edition of this chapter, and it includes modifications we have made over the past several years that further enhance transduction efficacy.

Abstract PO-041: Systemic screening of gene delivery methods in pancreatic ductal adenocarcinoma cells

Abstract Deaths in the United States due to Pancreatic Ductal Adenocarcinoma (PDAC) has risen steadily since 1990, and PDAC is expected to be the second leading cause of cancer death by 2025. Such a dismal prognosis is mainly attributed to the fact that tumors are detected too late for an effective treatment. Additionally, even when detected early, PDAC is challenging to treat with existing treatment options. Thus, there is a need for improved treatment strategies for pancreatic cancer patients. Great efforts have been made to develop and test new targets in various model systems such as tumor cell lines, tumor organoid models, and patient-derived xenografts (PDXs). We previously demonstrated a proof-of-principle of cellular reprogramming to pluripotent state to model PDAC progression and utilized it as a discovery tool for early detection markers for PDAC. However, reprogramming efficiency was remarkably low and it is, at least in part, attributed to uneven gene delivery efficiencies across cell types in human pancreatic normal and cancer cells. Intriguingly, classical ductal cell types were more resistant to transduction with conventional VSV-G-pseudotyped lentivirus (LeV) than squamous subtype pancreatic cancer. To better understand the susceptibility of different types of pancreatic cancer cells to viral transduction and improve the gene delivery efficiency, we analyzed transduction of LeV, Sendai Virus (SeV), and episomal vector transfection efficiencies in classical ductal and squamous cell types. We used human pancreatic duct epithelial and foreskin fibroblast cell lines as controls. We found that transduction efficiency of LeV in both types of PDAC cell lines are significantly lower compared to control cell lines and considerably higher in squamous type compared to classical ductal type. In contrast, transduction efficiency of SeV was similar for both classical ductal and squamous types of PDAC cell lines and significantly higher compared to LeV efficiency (90% vs 5-25% at MOI 1). We also found that nucleofection transfection efficiency of episomal vector is significantly higher in squamous cell type compared to classical ductal cells (67% vs 55%). This is significantly higher than transduction efficiency of LeV (25 and 5% at MOI 1 correspondingly) but lower compared to SeV transduction efficiency. Thus, both SeV and nucleofection delivery methods show higher efficiency compared to LeV and can be successfully used as gene delivery methods in PDAC cell lines. By completing this study, we can provide a tailored gene delivery method for pancreatic cancer, and this information can be harnessed for cellular reprograming of human PDAC as well as testing newly developing targets in other model systems in vitro and ex vivo.This work is supported by OHSU/CEDAR project award 68182-939-000. Citation Format: Dmytro Grygoryev, Taelor Ekstrom, Jason M. Link, Rosalie C. Sears, Jungsun Kim. Systemic screening of gene delivery methods in pancreatic ductal adenocarcinoma cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-041.

A Dedifferentiated Sinusoidal Endothelium Impacts Liver-Directed Gene Transfer in Hemophilia-a Mice

Hemophilia A is an X-linked recessive bleeding disorder that affects 1 in 5000 males and is caused by procoagulant factor VIII deficiency. Affected people are at danger of spontaneous bleeding into organs, which can be fatal and lead to persistent damage. Current therapy includes intravenous infusion of FVIII protein concentrate which carries the danger of transmitting blood-borne diseases. As a result of recent advancements in liver-directed gene transfer, gene therapy based innovative strategy for treating hemophilia has emerged. In patients with severe hemophilia B, intravenous infusion of an adeno-associated viral (AAV) vector encoding factor IX (FIX) under the control of a liver-directed promoter resulted in expression of FIX for a considerable period of time. In hemophilia-A patients, gene treatment utilizing AAV vectors has demonstrated to be less effective than Hemophilia B due to the size of the F8 coding sequence and the decreased release of FVIII protein. Among other concerns high immunogenicity of FVIII with 25-30% of hemophilia A patients forming inhibitors and overexpression of FVIII in hepatocytes triggering a cellular stress response are significantly challenging. A phase 1 clinical trial is now being conducted to examine the AAV8 induced liver directed gene expression strategy to circumvent these challenges.The Factor VIII null mouse has been effective in understanding the disease pathogenesis as well as the development of liver directed novel gene therapy techniques to treat hemophilia. FVIII is predominantly produced in the liver. Thus, liver directed adenoviral and retroviral vectors have been studied by several groups to understand the gene delivery method in hemophilia. A few of these studies have shown limited effectiveness in hemophilia animal models. Although the coagulation anomaly seen in hemophilia murine model was completely repaired immediately after liver directed adenovirus-mediated treatment, the effect was transient. Additionally, adeno associated virus (AAV8)-FVIII overexpression has been associated with increased cellular stress. In this study we evaluated the stability and efficacy of liver driven gene transfer mechanism in FVIII null mouse using recombinant AAV8 vector. Recombinant AAV8 vector delivered through the systemic circulation successfully transduces to target tissues via passing through the permeable barrier of sinusoidal endothelial cell. The bidirectional passage through sinusoidal endothelial cell is mainly supported by the presence of discontinuous fenestrated endothelium. Remarkably, we found that liver directed gene transfer was significantly delayed in FVIII null mice. Using quantitative liver intravital imaging we found that upon AAV8-GFP administration liver sinusoidal endothelial cells shows increased apoptosis. Moreover, structural analysis of the liver sinusoidal endothelial cells using intravital and electron micrograph imaging showed significant structural functional difference in liver sinusoidal endothelial cells of FVIII KO mouse. Work is currently underway to understand how absence of FVIII can affect the LSECs. In conclusion, detailed molecular characterization of LSEC-mediated liver directed gene transfer in a hemophilia mouse model is critical for understanding the efficacy and stability of gene-based hemophilia treatment. DisclosuresSundd: Bayer: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring Inc: Research Funding.

Human gene therapy: A patent analysis.

Although seen as a revolution in modern science, gene therapy has been plagued by failed clinical trials and controversial ethics in the last thirty years. Moreover, there is no comprehensive, in-depth, high-quality analysis of global gene therapy patents. This paper proposes a method to correctly retrieve patents to address the issue and use it for the patent landscape. The results show the global patent landscape of gene therapy, with the United States dominating the field, while China has emerged as a leader in recent years. For various reasons, the EU, Korea, and Japan lag in the development of patented technologies. China has edged closer to the US in both live and indefinite patents, with the Chinese Academy of Military Medical Sciences and the Chinese Academy of Sciences leading the way, surpassing primary applicants such as the US Department of Health and Human Services, the University of California, and the University of Pennsylvania. The study also reveals four broad categories of technologies that have been extensively studied in gene therapy: basic biology of the gene and diseases, diseases being treated, gene delivery methods, and potential adverse events. What is more, Adeno-Associated Virus, Retrovirus, and Lentivirus are the most prevalent gene therapy delivery vectors after 2014. The industrial development trend revealed in this paper can provide an evidence-based basis for scientific research management and decision-making.

Reduction of plasmid vector backbone length enhances reporter gene expression

Gene therapy has a wide range of applications for various types of pathologies. Viral methods of gene delivery provide high levels of gene expression but have various safety concerns. Non-viral methods are largely known to provide lower levels of expression. We aim to address this issue by using plasmid DNA with smaller backbones to increase gene expression levels when delivered using non-viral methods. In this study we compare gene expression levels between two vectors with firefly luciferase encoding gene insert using liposome complexes and gene electrotransfer as delivery methods. A 2-fold reduction in plasmid vector backbone size, disproportionately enhanced gene expression levels more than 10-fold in rat tenocytes in vitro, and rat myocardium in vivo, while improvements in delivery to the skin were more moderate.

Genetic interference exerted by Salmonella-delivered CRISPR/Cas9 significantly reduces the pathological burden caused by Marek\u2019s disease virus in chickens

Efficient in vivo delivery of a CRISPR/Cas9 plasmid is of paramount importance for effective therapy. Here, we investigated the usability of Salmonella as a plasmid carrier for in vivo therapy against virus-induced cancer using Marek’s disease virus (MDV) as a model for study in chickens. A green fluorescent protein-expressing CRISPR/Cas9 plasmid encoding the virulence gene pp38 was constructed against Marek’s disease virus. Therapeutic plasmids were transformed into Salmonella carrying lon and sifA gene deletions. The animals in 5 groups were intraperitoneally inoculated with phosphate-buffered saline, vector control, or Salmonella before or after MDV infection, or left uninfected as a naïve control. Therapeutic effectiveness was evaluated by observing disease outcomes and the viral copy number in peripheral blood mononuclear cells. The efficacy of plasmid delivery by Salmonella was 13 ± 1.7% in the spleen and 8.0 ± 1.8% in the liver on the 6th day post-infection. The Salmonella-treated groups showed significant resistance to MDV infection. The maximum effect was observed in the group treated with Salmonella before MDV infection. None of the chickens fully recovered; however, the results suggested that timely delivery of Salmonella could be effective for in vivo CRISPR/Cas9-mediated genetic interference against highly pathogenic MDV. The use of Salmonella in CRISPR systems provides a simpler and more efficient platform for in vivo therapy with CRISPR than the use of conventional in vivo gene delivery methods and warrants further development.

A guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system

Gene therapy revolves around modifying genetic makeup by inserting foreign nucleic acids into targeted cells via gene delivery methods to treat a particular disease. While the genes targeted play a key role in gene therapy, the gene delivery system used is also of utmost importance as it determines the success of gene therapy. As primary cells and stem cells are often the target cells for gene therapy in clinical trials, the delivery system would need to be robust, and viral-based entries such as lentiviral vectors work best at transporting the transgene into the cells. However, even within lentiviral vectors, several parameters can affect the functionality of the delivery system. Using cardiac-derived c-kit expressing cells (CCs) as a model system, this study aims to optimize lentiviral production by investigating various experimental factors such as the generation of the lentiviral system, concentration method, and type of selection marker. Our findings showed that the 2nd generation system with pCMV-dR8.2 dvpr as the packaging plasmid produced a 7.3-fold higher yield of lentiviral production compared to psPAX2. Concentrating the virus with ultracentrifuge produced a higher viral titer at greater than 5 × 105 infectious unit values/ml (IFU/ml). And lastly, the minimum inhibitory concentration (MIC) of puromycin selection marker was 10 μg/mL and 7 μg/mL for HEK293T and CCs, demonstrating the suitability of antibiotic selection for all cell types. This encouraging data can be extrapolated and applied to other difficult-to-transfect cells, such as different types of stem cells or primary cells.

Aluminum particles generated during millisecond electric pulse application enhance adenovirus-mediated gene transfer in L929 cells

Gene electrotransfer is an attractive method of non-viral gene delivery. However, the mechanism of DNA penetration across the plasma membrane is widely discussed. To explore this process for even larger structures, like viruses, we applied various combinations of short/long and high/low-amplitude electric pulses to L929 cells, mixed with a human adenovirus vector expressing GFP. We observed a transgene expression increase, both in the number of GFP-converted cells and GFP levels, when we added a low-voltage/millisecond-pulse treatment to the adenovirus/cell mixture. This increase, reflecting enhanced virus penetration, was proportional to the applied electric field amplitude and pulse number, but was not associated with membrane permeabilization, nor to direct cell modifications. We demonstrated that this effect is mainly due to adenovirus particle interactions with aggregated aluminum particles released from energized electrodes. Indeed, after centrifugation of the pulsed viral suspension and later on addition to cells, the activity was found mainly associated with the aluminum aggregates concentrated in the lower fraction and was proportional to generated quantities. Overall, this work focused on the use of electrotransfer to facilitate the adenovirus entry into cell, demonstrating that modifications of the penetrating agent can be more important than modifications of the target cell for transfer efficacy.

Biomaterial-based delivery of nucleic acids for tissue regeneration.

Gene therapy is a promising novel method of tissue regeneration by stimulating or inhibiting key signaling pathways. However, their therapeutic applications in vivo are largely limited by several physiological obstacles, such as degradation of nucleases, impermeability of cell membranes, and transport to the desired intracellular compartments. Biomaterial-based gene delivery systems can overcome the problems of stability and local drug delivery, and can temporarily control the overexpression of therapeutic genes, leading to the local production of physiologically relevant levels of regulatory factors. But the gene delivery of biomaterials for tissue regeneration relies on multi-factor design. This review aims to outline the impact of gene delivery methods, therapeutic genes and biomaterials selection on this strategy, emphatically introduce the latest developments in the design of gene delivery vehicles based on biomaterials, summarize the mechanism of nucleic acid for tissue regeneration, and explore the strategies of nucleic acid delivery vehicles for various tissue regeneration.

Recent Advances in Gene Therapy for Cardiac Tissue Regeneration.

Cardiovascular diseases (CVDs) are responsible for enormous socio-economic impact and the highest mortality globally. The standard of care for CVDs, which includes medications and surgical interventions, in most cases, can delay but not prevent the progression of disease. Gene therapy has been considered as a potential therapy to improve the outcomes of CVDs as it targets the molecular mechanisms implicated in heart failure. Cardiac reprogramming, therapeutic angiogenesis using growth factors, antioxidant, and anti-apoptotic therapies are the modalities of cardiac gene therapy that have led to promising results in preclinical studies. Despite the benefits observed in animal studies, the attempts to translate them to humans have been inconsistent so far. Low concentration of the gene product at the target site, incomplete understanding of the molecular pathways of the disease, selected gene delivery method, difference between animal models and humans among others are probable causes of the inconsistent results in clinics. In this review, we discuss the most recent applications of the aforementioned gene therapy strategies to improve cardiac tissue regeneration in preclinical and clinical studies as well as the challenges associated with them. In addition, we consider ongoing gene therapy clinical trials focused on cardiac regeneration in CVDs.

Advances in Gene Delivery Methods to Label and Modulate Activity of Upper Motor Neurons: Implications for Amyotrophic Lateral Sclerosis.

The selective degeneration of both upper motor neurons (UMNs) and lower motor neurons (LMNs) is the pathological hallmark of amyotrophic lateral sclerosis (ALS). Unlike the simple organisation of LMNs in the brainstem and spinal cord, UMNs are embedded in the complex cytoarchitecture of the primary motor cortex, which complicates their identification. UMNs therefore remain a challenging neuronal population to study in ALS research, particularly in the early pre-symptomatic stages of animal models. A better understanding of the mechanisms that lead to selective UMN degeneration requires unequivocal visualization and cellular identification of vulnerable UMNs within the heterogeneous cortical neuronal population and circuitry. Here, we review recent novel gene delivery methods developed to cellularly identify vulnerable UMNs and modulate their activity in various mouse models. A critical overview of retrograde tracers, viral vectors encoding reporter genes and transgenic reporter mice used to visualize UMNs in mouse models of ALS is provided. Functional targeting of UMNs in vivo with the advent of optogenetic and chemogenetic technology is also discussed. These exciting gene delivery techniques will facilitate improved anatomical mapping, cell-specific gene expression profiling and targeted manipulation of UMN activity in mice. These advancements in the field pave the way for future work to uncover the precise role of UMNs in ALS and improve future therapeutic targeting of UMNs.

Retrograde Parotid Gland Infusion through Stensen's Duct in a Non-Human Primate for Vectored Gene Delivery.

Salivary glands are an attractive tissue target for gene therapy with promising results already leading to human trials. They are inherently capable of secreting proteins into the bloodstream and are easily accessible, making them potentially superior tissue sites for replacement hormone production or vaccination by gene transfer. Suggested methods for gene delivery include transcutaneous injection and retrograde infusion through salivary ducts. We demonstrate how to perform Retrograde Salivary Gland Infusion (RSGI) in non-human primates. We describe the important anatomic landmarks including identification of the parotid papilla, an atraumatic method of cannulating and sealing Stensen's Duct utilizing basic dental tools, polyethylene tubing, and cyanoacrylate, and the appropriate rate of infusion. While this is the least traumatic method of delivery, the method is still limited by the volume able to be delivered (<0.5 mL) and the potential for trauma to the duct and gland. We demonstrate using fluoroscopy that an infusate can be fully delivered into the gland, and further demonstrate by immunohistochemistry the transduction of a typical vector and expression of the delivered gene.

High-throughput evaluation of polymeric nanoparticles for tissue-targeted gene expression using barcoded plasmid DNA

Successful systemic gene delivery requires specific tissue targeting as well as efficient intracellular transfection. Increasingly, research laboratories are fabricating libraries of novel nanoparticles, engineering both new biomaterial structures and composition ratios of multicomponent systems. Yet, methods for screening gene delivery vehicles directly in vivo are often low-throughout, limiting the number of candidate nanoparticles that can be investigated. Here, we report a comprehensive, high-throughput method to evaluate a library of polymeric nanoparticles in vivo for tissue-specific gene delivery. The method involves pairing each nanoparticle formulation with a plasmid DNA (pDNA) that harbors a unique nucleotide sequence serving as the identifying “barcode”. Using real time quantitative PCR (qPCR) for detection of the barcoded pDNA and quantitative reverse transcription PCR (RT-qPCR) for transcribed barcoded mRNA, we can quantify accumulation and transfection in tissues of interest. The barcode pDNA and primers were designed with sufficient sensitivity and specificity to evaluate multiple nanoparticle formulations per mouse, improving screening efficiency. Using this platform, we evaluated the biodistribution and transfection of 8 intravenously administered poly(beta-amino ester; PBAE) nanoparticle formulations, each with a PBAE polymer of differential structure. Significant levels of nanoparticle accumulation and gene transfection were observed mainly in organs involved in clearance, including spleen, liver, and kidneys. Interestingly, higher levels of transfection of select organs did not necessarily correlate with higher levels of tissue accumulation, highlighting the importance of directly measuring in vivo transfection efficiency as the key barcoded parameter in gene delivery vector optimization. To validate this method, nanoparticle formulations were used individually for luciferase pDNA delivery in vivo. The distribution of luciferase expression in tissues matched the transfection analysis by the barcode qPCR method, confirming that this platform can be used to accurately evaluate systemic gene delivery.

Real-time impedance feedback to enhance cutaneous gene electrotransfer in a murine skin model

Electric field mediated gene delivery methods have the ability to efficiently transfect cells in vivo with an excellent safety profile. The method has historically used a fixed number of electric pulses with identical characteristics in induce delivery. Electrical treatment does not typically compensate for subject-to-subject variation and other differences. This study was designed to investigate if delivery/expression could be increased using a novel electropulsation method that compensated for variation using real-time electrical impedance measurements. The method involved delivering plasmid DNA encoding luciferase to murine skin. Tissue impedance in a 1–3 KHz range was measured before electric pulses were applied. Impedance was also measured after each successive pulse. Pulsation was stopped when impedance values were reduced by either 80% or 95% relative to prepulse values. Standard/fixed pulsing parameters were also used for comparison. The results indicated that up to 15-fold increases in luciferase expression could be obtained when electrical treatment was ceased based upon impedance reductions. Furthermore, peak expression levels of all treatment groups pulsed using the novel pulsing method were statistically higher than those that employed standard pulsing. These results strongly suggest that applying pulses until a defined impedance-based endpoint results in higher expression.