Sonoselective delivery using ultrasound and microbubbles combined with intravenous rAAV9 CLDN5-GFP does not increase endothelial gene expression.

Transcranial ultrasound combined with intravenous microbubbles can be used to increase blood-brain barrier permeability or, at lower pressures, to mediate sonoselective gene delivery to endothelial cells. Previously, sonoselective gene delivery with plasmid-coated microbubbles as gene carriers resulted in transient transgene expression in the brain endothelium. We investigated the potential of recombinant adeno-associated virus 9 (rAAV9), a serotype known for its efficient transduction and long-term transgene expression, for sonoselective gene delivery to endothelial cells of the brain. We found that rAAV9 led to gene delivery to brain endothelial cells following intravenous administration at a dosage of 1 × 1011 GC/g. However, the sonoselective gene delivery approach with intravenous rAAV9, using the same parameters as previously used for plasmid delivery, did not increase transgene expression in brain endothelial cells targeted. These results suggest that intravenous rAAV9 are using mechanisms of entry into the cerebrovasculature that are not significantly influenced by sonoselective treatments known to facilitate endothelial cell entry of plasmids coated onto microbubbles.

Reference standards for accurate validation and optimization of assays that determine integrated lentiviral vector copy number in transduced cells.

Lentiviral vectors (LV) have emerged as a robust technology for therapeutic gene delivery into human cells as advanced medicinal products. As these products are increasingly commercialized, there are concomitant demands for their characterization to ensure safety, efficacy and consistency. Standards are essential for accurately measuring parameters for such product characterization. A critical parameter is the vector copy number (VCN) which measures the genetic dose of a transgene present in gene-modified cells. Here we describe a set of clonal Jurkat cell lines with defined copy numbers of a reference lentiviral vector integrated into their genomes. Genomic DNA was characterized for copy number, genomic integrity and integration coordinates and showed uniform performance across independent quantitative PCR assays. Stability studies during continuous long-term culture demonstrated sustained renewability of the reference standard source material. DNA from the Jurkat VCN standards would be useful for control of quantitative PCR assays for VCN determination in LV gene-modified cellular products and clinical samples.

Optimization of adenoviral gene transfer in human pluripotent stem cells.

Human pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, have the potential to differentiate into a wide variety of cells in vitro and have applications in basic developmental biology research and regenerative medicine. To understand the process of differentiation from pluripotent stem cells to functional cells, it is necessary to efficiently and safely transfer and express exogenous genes. We attempted to optimize the efficient transfer of genes into pluripotent stem cells using adenoviral vectors. Comparative study of the activities of three representative ubiquitously active promoters revealed that only the CA promoter allowed robust transgene expression in human pluripotent stem cells. In addition, we established a protocol that allowed us to efficiently introduce target genes and ensure their expression even in small numbers of cells. Adenoviral vector infection of pluripotent stem cells in single-cell suspension culture yielded high gene transfer efficiency with low cytotoxicity, without losing the undifferentiated state of the pluripotent stem cells. This optimized system will facilitate developmental biology research and regenerative medicine using pluripotent stem cells.

High-Throughput Automation of Endosomolytic Polymers for mRNA Delivery.

In recent years, there has been an increasing interest in designing delivery systems to enhance the efficacy of RNA-based therapeutics. Here, we have synthesized copolymers comprised of dimethylaminoethyl methacrylate (DMAEMA) or diethylaminoethyl methacrylate (DEAEMA) copolymerized with alkyl methacrylate monomers ranging from 2 to 12 carbons, and developed a high throughput workflow for rapid investigation of their applicability for mRNA delivery. The structure activity relationship revealed that the mRNA encapsulation efficiency is improved by increasing the cationic density and use of shorter alkyl side chains (2-6 carbons). Minimal cytotoxicity was observed when using DEAEMA-co-BMA (EB) polyplexes up to 18 h after dosing, independent of a poly(ethylene glycol) (PEG) first block. The lowest molecular weight polymer (EB10,250) performed best, exhibiting greater transfection than polyethyenimine (PEI) based upon the number of cells transfected and mean intensity. Conventional investigations into the performance of polymeric materials for mRNA delivery is quite tedious, consequently limiting the number of materials and formulation conditions that can be studied. The high throughput approach presented here can accelerate the screening of polymeric systems and paves the way for expanding this generalizable approach to assess various materials for mRNA delivery.

GMP-Compliant Adenoviral Vectors for Gene Therapy.

Recently, gene therapy as one of the most promising treatments can apply genes for incurable diseases treatment. In this context, vectors as gene delivery systems play a pivotal role in gene therapy procedure. Hereupon, viral vectors have been increasingly introduced as a hyper-efficient tools for gene therapy. Adenoviral vectors as one of the most common groups which are used in gene therapy have a high ability for humans. Indeed, they are not integrated into host genome. In other words, they can be adapted for direct transduction of recombinant proteins into targeted cells. Moreover, they have large packaging capacity and high levels of efficiency and expression. In accordance with translational pathways from the basic to the clinic, recombinant adenoviral vectors packaging must be managed under good manufacturing practice (GMP) principles before applying in clinical trials. Therein, in this chapter standard methods for manufacturing of GMP-compliant Adenoviral vectors for gene therapy have been introduced.

Gene Delivery to the Skin - How Far Have We Come?

Gene therapies are powerful tools to prevent, treat, and cure human diseases. The application of gene therapies for skin diseases received little attention so far, despite the easy accessibility of skin and the urgent medical need. A major obstacle is the unique barrier properties of human skin, which significantly limits the absorption of biomacromolecules, and thus hampers the efficient delivery of nucleic acid payloads. In this review, we discuss current approaches, successes, and failures of cutaneous gene therapy and provide guidance toward the development of next-generation concepts. We specifically allude to the delivery strategies as the major obstacle that prevents the full potential of gene therapies - not only for skin disorders but also for almost any other human disease.

Human-derived NLS enhance the gene transfer efficiency of chitosan.

Nuclear import is considered as one of the major limitations for non-viral gene delivery systems and the incorporation of nuclear localization signals (NLS) that mediate nuclear intake can be used as a strategy to enhance internalization of exogenous DNA. In this work, human-derived endogenous NLS peptides based on insulin growth factor binding proteins (IGFBP), namely IGFBP-3 and IGFBP-5, were tested for their ability to improve nuclear translocation of genetic material by non-viral vectors. Several strategies were tested to determine their effect on chitosan mediated transfection efficiency: co-administration with polyplexes, co-complexation at the time of polyplex formation, and covalent ligation to chitosan. Our results show that co-complexation and covalent ligation of the NLS peptide derived from IGFBP-3 to chitosan polyplexes yields a 2-fold increase in transfection efficiency, which was not observed for NLS peptide derived from IGFBP-5. These results indicate that the integration of IGFBP-NLS-3 peptides into polyplexes has potential as a strategy to enhance the efficiency of non-viral vectors.

Ultrasound-assisted magnetic nanoparticle-based gene delivery.

Targeted gene delivery is important in biomedical research and applications. In this paper, we synergistically combine non-viral chemical materials, magnetic nanoparticles (MNPs), and a physical technique, low-intensity pulsed ultrasound (LIPUS), to achieve efficient and targeted gene delivery. The MNPs are iron oxide super-paramagnetic nanoparticles, coated with polyethyleneimine (PEI), which makes a high positive surface charge and is favorable for the binding of genetic materials. Due to the paramagnetic properties of the MNPs, the application of an external magnetic field increases transfection efficiency while LIPUS stimulation enhances cell viability and permeability. We found that stimulation at the intensity of 30 mW/cm2 for 10 minutes yields optimal results with a minimal adverse effect on the cells. By combining the effect of the external magnetic field and LIPUS, the genetic material (GFP or Cherry Red plasmid) can enter the cells. The flow cytometry results showed that by using just a magnetic field to direct the genetic material, the transfection efficiency on HEK 293 cells that were treated by our MNPs was 56.1%. Coupled with LIPUS stimulation, it increased to 61.5% or 19% higher than the positive control (Lipofectamine 2000). Besides, compared with the positive control, our method showed less toxicity. Cell viability after transfection was 63.61%, which is 19% higher than the standard transfection technique. In conclusion, we designed a new gene-delivery method that is affordable, targeted, shows low-toxicity, yet high transfection efficiency, compared to other conventional approaches.

Delivery Approaches for Therapeutic Genome Editing and Challenges.

Impressive therapeutic advances have been possible through the advent of zinc-finger nucleases and transcription activator-like effector nucleases. However, discovery of the more efficient and highly tailorable clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas9) has provided unprecedented gene-editing capabilities for treatment of various inherited and acquired diseases. Despite recent clinical trials, a major barrier for therapeutic gene editing is the absence of safe and effective methods for local and systemic delivery of gene-editing reagents. In this review, we elaborate on the challenges and provide practical considerations for improving gene editing. Specifically, we highlight issues associated with delivery of gene-editing tools into clinically relevant cells.

Gene therapy: a double-edged sword with great powers.

Gene therapy is the treatment of a disease through transferring genetic material into cells of the patients. In the recent several years, gene therapy has experienced rapid progress and achieved huge success. Over two dozens of gene therapies have been approved for clinical use by the drug regulatory agencies from different countries. However, concerns about its efficacy and safety have accompanied gene therapy since its birth. In the present manuscript, we first introduce various strategies employed in gene therapy, which includes ex vivo gene delivery v.s. in vivo gene delivery; gene addition v.s. genome editing; inherited disease v.s. acquired disease; and somatic gene therapy v.s. germline gene therapy. Then we discuss the clinical outcomes of some approved gene therapies. We finish our discussion with the safety issues related to gene therapy. We will see that with the technology improvement, somatic gene therapy has been proved to be efficient and safe enough for clinical practice. However, germline gene therapy has important efficiency and safety issues at present, and should not be put into clinical practice before these issues are solved.

Immunoadsorption enables successful rAAV5-mediated repeated hepatic gene delivery in nonhuman primates.

Adeno-associated virus (AAV)-based liver gene therapy has been shown to be clinically successful. However, the presence of circulating neutralizing antibodies (NABs) against AAV vector capsids remains a major challenge as it may prevent successful transduction of the target cells. Therefore, there is a need to develop strategies that would enable AAV-mediated gene delivery to patients with preexisting anti-AAV NABs. In the current study, the feasibility of using an immunoadsorption (IA) procedure for repeated, liver-targeted gene delivery in nonhuman primates was explored. The animals were administered IV with recombinant AAV5 (rAAV5) carrying the reporter gene human secreted embryonic alkaline phosphatase (hSEAP). Seven weeks after the first rAAV treatment, all of the animals were readministered with rAAV5 carrying the therapeutic hemophilia B gene human factor IX (hFIX). Half of the animals administered with rAAV5-hSEAP underwent IA prior to the second rAAV5 exposure. The transduction efficacies of rAAV5-hSEAP and rAAV5-hFIX were assessed by measuring the levels of hSEAP and hFIX proteins. Although no hFIX was detected after rAAV5-hFIX readministration without prior IA, all animals submitted to IA showed therapeutic levels of hFIX expression, and a threshold of anti-AAV5 NAB levels compatible with successful readministration was demonstrated. In summary, our data demonstrate that the use of a clinically applicable IA procedure enables successful readministration of an rAAV5-based gene transfer in a clinically relevant animal model. Finally, the analysis of anti-AAV NAB levels in human subjects submitted to IA confirmed the safety and efficacy of the procedure to reduce anti-AAV NABs. Furthermore, clinical translation was assessed using an immunoglobulin G assay as surrogate.

The Impact of Alkyl-Chain Purity on Lipid-Based Nucleic Acid Delivery Systems - Is the Utilization of Lipid Components with Technical Grade Justified?

The physicochemical properties and transfection efficacies of two samples of a cationic lipid have been investigated and compared in 2D (monolayers at the air/liquid interface) and 3D (aqueous bulk dispersions) model systems using different techniques. The samples differ only in their chain composition due to the purity of the oleylamine (chain precursor). Lipid 8 (using the oleylamine of technical grade for cost-efficient synthesis) shows lateral phase separation in the Langmuir layers. However, the amount of attached DNA, determined by IRRAS, is for both samples the same. In 3D systems, lipid 8 p forms cubic phases, which disappear after addition of DNA. At physiological temperatures, both lipids (alone and in mixture with cholesterol) assemble to lamellar aggregates and exhibit comparable DNA delivery efficiency. This study demonstrates that non-lamellar structures are not compulsory for high transfection rates. The results legitimate the utilization of oleyl chains of technical grade in the synthesis of cationic transfection lipids.

Evaluation of AAV-mediated delivery of shRNA to target basal-like breast cancer genetic vulnerabilities.

Adeno-associated viral vectors (AAV) for gene therapy applications are gaining momentum, with more therapies moving into later stages of clinical development and towards market approval, namely for cancer therapy. The development of cytotoxic vectors is often hampered by side effects arising when non-target cells are infected, and their production can be hindered by toxic effects of the transgene on the producing cell lines. In this study, we evaluated the potential of rAAV-mediated delivery of short hairpin RNAs (shRNA) to target basal-like breast cancer genetic vulnerabilities. Our results show that by optimizing the stoichiometry of the plasmids upon transfection and time of harvest, it is possible to increase the viral titers and quality. All rAAV-shRNA vectors obtained efficiently transduced the BLBC cell lines MDA-MB-468 and HCC1954. In MDA-MB-468, transduction with rAAV-shRNA vector targeting PSMA2 was associated with significant decrease in cell viability and apoptosis induction. Importantly, rAAV2-PSMA2 also slowed tumor growth in a BLBC mouse xenograft model, thus potentially representing a therapeutic strategy against this type of cancer.

A Critical Review of Electroporation as A Plasmid Delivery System in Mouse Skeletal Muscle.

The gene delivery to skeletal muscles is a promising strategy for the treatment of both muscular disorders (by silencing or overexpression of specific gene) and systemic secretion of therapeutic proteins. The use of a physical method like electroporation with plate or needle electrodes facilitates long-lasting gene silencing in situ. It has been reported that electroporation enhances the expression of the naked DNA gene in the skeletal muscle up to 100 times and decreases the changeability of the intramuscular expression. Coelectransfer of reporter genes such as green fluorescent protein (GFP), luciferase or beta-galactosidase allows the observation of correctly performed silencing in the muscles. Appropriate selection of plasmid injection volume and concentration, as well as electrotransfer parameters, such as the voltage, the length and the number of electrical pulses do not cause long-term damage to myocytes. In this review, we summarized the electroporation methodology as well as the procedure of electrotransfer to the gastrocnemius, tibialis, soleus and foot muscles and compare their advantages and disadvantages.

Efficient gene transfer into T lymphocytes by fiber-modified human adenovirus 5.

BACKGROUND: The gene transduction efficiency of adenovirus to hematopoietic cells, especially T lymphocytes, is needed to be improved. The purpose of this study is to improve the transduction efficiency of T lymphocytes by using fiber-modified human adenovirus 5 (HAdV-5) vectors. RESULTS: Four fiber-modified human adenovirus 5 (HAdV-5) vectors were investigated to transduce hematopoietic cells. F35-EG or F11p-EG were HAdV-35 or HAdV-11p fiber pseudotyped HAdV-5, and HR-EG or CR-EG vectors were generated by incorporating RGD motif to the HI loop or to the C-terminus of F11p-EG fiber. All vectors could transduce more than 90% of K562 or Jurkat cells at an multiplicity of infection (MOI) of 500 viral particle per cell (vp/cell). All vectors except HR-EG could transduce nearly 90% cord blood CD34+ cells or 80% primary human T cells at the MOI of 1000, and F11p-EG showed slight superiority to F35-EG and CR-EG. Adenoviral vectors transduced CD4+ T cells a little more efficiently than they did to CD8+ T cells. These vectors showed no cytotoxicity at an MOI as high as 1000 vp/cell because the infected and uninfected T cells retained the same CD4/CD8 ratio and cell growth rate. CONCLUSIONS: HAdV-11p fiber pseudotyped HAdV-5 could effectively transduce human T cells when human EF1a promoter was used to control the expression of transgene, suggesting its possible application in T cell immunocellular therapy.

Comprehensive evaluation of ubiquitous promoters suitable for the generation of transgenic cynomolgus monkeys†.

Nonhuman primates (NHPs) are considered to be the most valuable models for human transgenic (Tg) research into disease because human pathology is more closely recapitulated in NHPs than rodents. Previous studies have reported the generation of Tg NHPs that ubiquitously overexpress a transgene using various promoters, but it is not yet clear which promoter is most suitable for the generation of NHPs overexpressing a transgene ubiquitously and persistently in various tissues. To clarify this issue, we evaluated four putative ubiquitous promoters, cytomegalovirus (CMV) immediate-early enhancer and chicken beta-actin (CAG), elongation factor 1α (EF1α), ubiquitin C (UbC), and CMV, using an in vitro differentiation system of cynomolgus monkey embryonic stem cells (ESCs). While the EF1α promoter drove Tg expression more strongly than the other promoters in undifferentiated pluripotent ESCs, the CAG promoter was more effective in differentiated cells such as embryoid bodies and ESC-derived neurons. When the CAG and EF1α promoters were used to generate green fluorescent protein (GFP)-expressing Tg monkeys, the CAG promoter drove GFP expression in skin and hematopoietic tissues more strongly than in ΕF1α-GFP Tg monkeys. Notably, the EF1α promoter underwent more silencing in both ESCs and Tg monkeys. Thus, the CAG promoter appears to be the most suitable for ubiquitous and stable expression of transgenes in the differentiated tissues of Tg cynomolgus monkeys and appropriate for the establishment of human disease models.

Pharmaceutical Development of AAV-Based Gene Therapy Products for the Eye.

A resurgence of interest and investment in the field of gene therapy, driven in large part by advances in viral vector technology, has recently culminated in United States Food and Drug Administration approval of the first gene therapy product targeting a disease caused by mutations in a single gene. This product, LUXTURNA™ (voretigene neparvovec-rzyl; Spark Therapeutics, Inc., Philadelphia, PA), delivers a normal copy of the RPE65 gene to retinal cells for the treatment of biallelic RPE65 mutation-associated retinal dystrophy, a blinding disease. Many additional gene therapy programs targeting both inherited retinal diseases and other ocular diseases are in development, owing to an improved understanding of the genetic basis of ocular disease and the unique properties of the ocular compartment that make it amenable to local gene therapy. Here we review the growing body of literature that describes both the design and development of ocular gene therapy products, with a particular emphasis on target and vector selection, and chemistry, manufacturing, and controls.

Synthesis, characterization and evaluation of transfection efficiency of dexamethasone conjugated poly(propyleneimine) nanocarriers for gene delivery#.

CONTEXT: Polypropylenimine (PPI), a cationic dendrimer with defined structure and positive surface charge, is a potent non-viral vector. Dexamethasone (Dexa) conveys to the nucleus through interaction with its intracellular receptor. OBJECTIVE: This study develops efficient and non-toxic gene carriers through conjugation of Dexa at various percentages (5, 10 and 20%) to the fourth and the fifth generation PPIs (PPIG4s and PPIG5s). MATERIALS AND METHODS: The 21-OH group of Dexa (0.536 mmol) was modified with methanesulfonyl chloride (0.644 mmol) to activate it (Dexa-mesylate), and then it was conjugated to PPIs using Traut's reagent. After dialysis (48 h) and lyophilization, the physicochemical characteristics of products (PPI-Dexa) including zeta potential, size, buffering capacity and DNA condensing capability were investigated and compared with unmodified PPIs. Moreover, the cytotoxicity and transfection activity of the Dexa-modified PPIs were assessed using Neuro2A cells. RESULTS: Transfection of PPIG4 was close to PEI 25 kDa. Although the addition of Dexa to PPIG4s did not improve their transfection, their cytotoxicity was improved; especially in the carrier to DNA weight ratios (C/P) of one and two. The Dexa conjugation to PPIG5s enhanced their transfection at C/P ratio of one in both 5% (1.3-fold) and 10% (1.6-fold) Dexa grafting, of which the best result was observed in PPIG5-Dexa 10% at C/P ratio of one. DISCUSSION AND CONCLUSIONS: The modification of PPIs with Dexa is a promising approach to improve their cytotoxicity and transfection. The higher optimization of physicochemical characteristics, the better cell transfection and toxicity will be achieved.

Systemic Gene Delivery by Single-Dose Intracardiac Administration of scAAV2/9 and scAAV2/rh10 Variants in Newborn Rats.

Recombinant adeno-associated virus serotype 9 (rAAV2/9) and pseudotype rhesus-10 (rAAV2/rh10) are used for gene delivery, especially into the central nervous system. Both serotypes cross the blood-brain barrier and mediate stable long-term transduction in dividing and nondividing cells. Among possible routes of administration, intracardiac injection holds the potential for widespread vector diffusion associated with a relatively simple approach. In this study adopting the intracardiac route, we compare the cell-specific tropism and transfection efficacy of a panel of engineered rAAV2/9 and rAAV2/rh10 vectors encoding the enhanced green fluorescent protein. We observed transduction in the brain and peripherally, with a predominant neuronal tropism while the various serotypes achieved different expression patterns.

Optimized delivery of siRNA into 3D tumor spheroid cultures in situ.

3D tissue culture provides a physiologically relevant and genetically tractable system for studying normal and malignant human tissues. Despite this, gene-silencing studies using siRNA has proved difficult. In this study, we have identified a cause for why traditional siRNA transfection techniques are ineffective in eliciting gene silencing in situ within 3D cultures and proposed a simple method for significantly enhancing siRNA entry into spheroids/organoids. In 2D cell culture, the efficiency of gene silencing is significantly reduced when siRNA complexes are prepared in the presence of serum. Surprisingly, in both 3D tumour spheroids and primary murine organoids, the presence of serum during siRNA preparation rapidly promotes entry and internalization of Cy3-labelled siRNA in under 2 hours. Conversely, siRNA prepared in traditional low-serum transfection media fails to gain matrigel or spheroid/organoid entry. Direct measurement of CTNNB1 mRNA (encoding ß-catenin) from transfected tumour spheroids confirmed a transient but significant knockdown of ß-catenin when siRNA:liposome complexes were formed with serum, but not when prepared in the presence of reduced-serum media (Opti-MEM). Our studies suggest a simple modification to standard lipid-based transfection protocols facilitates rapid siRNA entry and transient gene repression, providing a platform for researchers to improve siRNA efficiency in established 3D cultures.