Increased Gene Transfer Research Articles

Hyperthermia Enhances Adeno-Associated Virus Vector Transduction Efficiency in Melanoma Cells.

Gene therapy perfectly fits in the current needs of medicine for patients with melanoma. One of the major challenges of gene therapy is to increase gene transfer. The role of hyperthermia in the improvement of AAV (adeno-associated virus) transduction efficiency has been indicated. The aim of the present study was to assess the transduction efficacy of melanoma cell lines (A375, G-361, and SK-MEL-1) with the use of the rAAV/DJ mosaic vector under hyperthermia conditions. The analysis of changes in the transduction efficacy and expression of HSPs (heat shock proteins) and receptors for AAV was performed. The transduction was performed at 37 °C and at 43 °C (1 h). Hyperthermia enhanced gene transfer in all the tested cell lines. The most efficient transducing cell line under hyperthermia was A375 (increase by 17%). G361 and SK-MEL-1 cells showed an increase of 7%. The changes in the expression of the AAV receptors and HSPs after hyperthermia were observed. A key role in the improvement of gene transfer may be played by AAVR, HSPB1, HSP6, DNAJC4, HSPD1, HSPA8, HSPA9, HSP90AB1, and AHSA1. This study showed the possibility of the use of hyperthermia as a factor enabling the stimulation of cell transduction with rAAV vectors, thereby providing tools for the improvement in the efficacy of gene therapy based on rAAV.

Phthalates Promote Dissemination of Antibiotic Resistance Genes: An Overlooked Environmental Risk.

Plastics-microorganism interactions have aroused growing environmental and ecological concerns. However, previous studies concentrated mainly on the direct interactions and paid little attention to the ecotoxicology effects of phthalates (PAEs), a common plastic additive that is continuously released and accumulates in the environment. Here, we provide insights into the impacts of PAEs on the dissemination of antibiotic resistance genes (ARGs) among environmental microorganisms. Dimethyl phthalate (DMP, a model PAE) at environmentally relevant concentrations (2-50 μg/L) significantly boosted the plasmid-mediated conjugation transfer of ARGs among intrageneric, intergeneric, and wastewater microbiota by up to 3.82, 4.96, and 4.77 times, respectively. The experimental and molecular dynamics simulation results unveil a strong interaction between the DMP molecules and phosphatidylcholine bilayer of the cell membrane, which lowers the membrane lipid fluidity and increases the membrane permeability to favor transfer of ARGs. In addition, the increased reactive oxygen species generation and conjugation-associated gene overexpression under DMP stress also contribute to the increased gene transfer. This study provides fundamental knowledge of the PAE-bacteria interactions to broaden our understanding of the environmental and ecological risks of plastics, especially in niches with colonized microbes, and to guide the control of ARG environmental spreading.

Relaxin-encapsulated polymeric metformin nanoparticles remodel tumor immune microenvironment by reducing CAFs for efficient triple-negative breast cancer immunotherapy

Cancer-associated fibroblasts (CAFs) are one of the most abundant stromal cells in the tumor microenvironment which mediate desmoplastic response and are the primary driver for an immunosuppressive microenvironment, leading to the failure of triple-negative breast cancer (TNBC) immunotherapy. Therefore, depleting CAFs may enhance the effect of immunotherapy (such as PD-L1 antibody). Relaxin (RLN) has been demonstrated to significantly improve transforming growth factor-β (TGF-β) induced CAFs activation and tumor immunosuppressive microenvironment. However, the short half-life and systemic vasodilation of RLN limit its in vivo efficacy. Here, plasmid encoding relaxin (pRLN) to locally express RLN was delivered with a new positively charged polymer named polymeric metformin (PolyMet), which could increase gene transfer efficiency significantly and have low toxicity that have been certified by our lab before. In order to improve the stability of pRLN in vivo, this complex was further formed lipid poly-γ-glutamic acid (PGA)/PolyMet-pRLN nanoparticle (LPPR). The particle size of LPPR was 205.5 ± 2.9 nm, and the zeta potential was +55.4 ± 1.6 mV. LPPR displayed excellent tumor penetrating efficacy and weaken proliferation of CAFs in 4T1luc/CAFs tumor spheres in vitro. In vivo, it could reverse aberrantly activated CAFs by decreasing the expression of profibrogenic cytokine and remove the physical barrier to reshape the tumor stromal microenvironment, which enabled a 2.2-fold increase in cytotoxic T cell infiltration within the tumor and a decrease in immunosuppressive cells infiltration. Thus, LPPR was observed retarded tumor growth by itself in the 4T1 tumor bearing-mouse, and the reshaped immune microenvironment further led to facilitate antitumor effect when it combined with PD-L1 antibody (aPD-L1). Altogether, this study presented a novel therapeutic approach against tumor stroma using LPPR to achieve a combination regimen with immune checkpoint blockade therapy against the desmoplastic TNBC model.

DNA Gyrase Inhibitors Increase the Frequency of Bacteriophage-like RcGTA-Mediated Gene Transfer in Rhodobacter capsulatus.

Rhodobacter capsulatus produces a bacteriophage-like particle called the gene transfer agent (RcGTA) that mediates horizontal gene transfer. RcGTA particles transfer random ~4.5-kb fragments of genomic DNA that integrate into recipient genomes by allelic replacement. This work addresses the effect of sub-inhibitory concentrations of antibiotics on gene transfer by RcGTA. A transduction assay was developed to test the effects of various substances on gene transfer. Using this assay, low concentrations of DNA gyrase inhibitors were found to increase the frequency of gene transfer. Novobiocin was studied in more detail, and it was found that this antibiotic did not influence the production or release of RcGTA but instead appeared to act on the recipient cells. The target of novobiocin in other species has been shown to be the GyrB subunit of DNA gyrase (a heterotetramer of 2GyrA and 2GyrB). R. capsulatus encodes GyrA and GyrB homologues, and a GyrB overexpression plasmid was created and found to confer resistance to novobiocin. The presence of the overexpression plasmid in recipient cells greatly diminished the novobiocin-mediated increase in gene transfer, confirming that this effect is due to the binding of novobiocin by GyrB. The results of this work show that antibiotics affect gene transfer in R. capsulatus and may be relevant to microbial genetic exchange in natural ecosystems.

Design and synthesis of multi-targeted nanoparticles for gene delivery to breast cancer tissues.

Biocompatibility of nanoparticles is the most essential factor in their use in clinical applications. In this study, hyperbranched spermine (HS), hyperbranched spermine-polyethylene glycol-folic acid (HSPF), and hyperbranched spermine-polyethylene glycol-glucose (HSPG) were synthesized for DNA protection and gene delivery to breast cancer cells. The synthesis of HSPG and HSPF was confirmed using proton nuclear magnetic resonance (H-NMR), Fourier-transform infrared spectroscopy (FTIR), and thermogravimetric analysis (TGA) spectroscopy. The HS/DNA, HSPF/DNA, HSPG/DNA, and hyperbranched spermine-polyethylene glycol-folic acid/glucose/DNA (HSPFG/DNA) nanoparticles were prepared by combining different concentrations of HS, HSPF, and HSPG with the same amount of DNA. The ability of HS, HSPF, and HSPG to interact with DNA and protect it against plasm digestion was evaluated using agarose gel. Moreover, in vivo and in vitro biocompatibility of HSPF/DNA, HSPG/DNA, and HSPFG/DNA was investigated using MTT assay and calculating weight change and survival ratio of BALB/c mice, respectively. The results of agarose gel electrophoresis showed that HS, HSPF, and HSPG have the high ability to neutralize the negative charge of DNA and protect it against plasma degradation. The results of in vivo cytotoxicity assay revealed that the HSPF/DNA, HSPG/DNA, and HSPFG/DNA nanoparticles have good biocompatibility on female BALB/c mice. In vitro and in vivo transfection assays revealed that functionalization of the surface of HS using polyethylene glycol-folic acid (HSPF) and polyethylene glycol-glucose (HSPG) significantly increases gene delivery efficiency in vitro and in vivo. These results also showed that gene transfer using both HSPF and HSPG copolymers increases gene transfer efficiency compared to when only one of them is used. The HSPFG/DNA nanoparticles have a high potential for use in therapeutic applications because of their excellent biocompatibility and high gene transfer efficiency to breast cancer tissue.

Mode of action of elasnin as biofilm formation eradicator of methicillin-resistant Staphylococcus aureus.

Biofilm is made up of microbes and their extracellular matrix, making microorganisms highly tolerant, resistant, and resilient to a wide range of antimicrobials. Biofilm treatment with conventional antimicrobial agents can accelerate the evolution and spread of resistance due to the reduced efficacy and increased gene transfer and differentiation within biofilms. Therefore, effective biofilm-targeting compounds are currently highly sought after. In the present study, we identified elasnin as a potent biofilm-targeting compound against methicillin-resistant Staphylococcus aureus (MRSA). Elasnin effectively inhibited biofilm formation and especially eradicated the pre-formed biofilms of MRSA with low cytotoxicity and low risk of resistance development and retains its activity in a chronic wound biofilms model. A comprehensive mechanistic study using multi-omics and confocal and scanning electron microscopy revealed that elasnin induced the biofilm matrix destruction in a time-dependent manner and interfered with the cell division during the exponential phase, primarily by repressing the expression of virulence factors. Cells released from the elasnin-treated biofilms exhibited a defective appearance and became more sensitive to beta-lactam antibiotic penicillin G. Through gene overexpression and deletion assay, we discovered the key role of sarZ during elasnin-induced biofilm eradication. Overall, the present study identified elasnin as a potent biofilm eradicator against MRSA that harbors potential to be developed for biofilm removal and chronic wound treatment, and provided new insights into the molecular targets for biofilm eradication in MRSA.

Manipulatingearly pig embryos

On the basis of established surgical procedures for embryo recovery and transfer, the early pig embryo can be subjected to various manipulations aimed at a longterm preservation of genetic material, the generation of identical multiplets, the early determination of sex or the alteration of the genetic make-up. Most of these procedures are still at an experimental stage and despite recent considerable progress are far from practical application. Normal piglets have been obtained after cryopreservation of pig blastocysts hatched in vitro, whereas all attempts to freeze embryos with intact zona pellucida have been unsuccessful. Pig embryos at the morula and blastocyst stage can be bisected microsurgically and the resulting demiembryos possess a high developmental potential in vitro, whereas their development in vivo is impaired. Pregnancy rates are similar (80%) but litter size is reduced compared with intact embryos and twinning rate is approximately 2%. Pig blastomeres isolated from embryos up to the 16-cell stage can be grown in culture and result in normal blastocysts. Normal piglets have been bom upon transfer of blastocysts derived from isolated eight-cell blastomeres, clearly underlining the totipotency of this developmental stage. Upon nuclear transfer the developmental capacity of reconstituted pig embryos is low and < 10% develop to morulae or blastocysts in vitro. Pig oocytes can be stimulated parthenogenetically and up to 10% grow to blastocysts in the in vitro culture. Sex determination can be achieved either by separation of X and Y chromosome bearing spermatozoa by flow cytometry or by analysing the expression of the HY antigen in pig embryos from the eight-cell to morula stage. Microinjection of foreign DNA has been successfully used to alter growth and development of transgenic pigs, and to produce foreign proteins in the mammary gland or in the bloodstream, indicating that pigs can be used as donors for valuable human pharmaceutical proteins. Another promising area of gene transfer is the increase of disease resistance in transgenic lines of pigs. Approximately 30% of pig spermatozoa bind considerable amounts of foreign DNA preferably at the post-acrosomal region, suggesting that transgenic animals can be obtained more efficiently than with the usual microinjection procedure. To increase gene transfer efficiency, considerable research efforts have been made to establish embryonic stem (ES) cells, but so far there is no definite proof of totipotency of the generated pig ESlike cells through viable chimaeras. In general, biotechnological procedures are much less advanced in pigs than in cows.

Coevolution of Adeno-associated Virus Capsid Antigenicity and Tropism through a Structure-Guided Approach.

Adeno-associated viruses (AAV) are composed of nonenveloped, icosahedral protein shells that can be adapted to package and deliver recombinant therapeutic DNA. Approaches to engineer recombinant capsids for gene therapy applications have focused on rational design or library-based approaches that can address one or two desirable attributes; however, there is an unmet need to comprehensively improve AAV vector properties. Such cannot be achieved by utilizing sequence data alone but requires harnessing the three-dimensional (3D) structural properties of AAV capsids. Here, we solve the structures of a natural AAV isolate complexed with antibodies using cryo-electron microscopy and harness this structural information to engineer AAV capsid libraries through saturation mutagenesis of different antigenic footprints. Each surface loop was evolved by infectious cycling in the presence of a helper adenovirus to yield a new AAV variant that then serves as a template for evolving the next surface loop. This stepwise process yielded a humanized AAV8 capsid (AAVhum.8) displaying nonnatural surface loops that simultaneously display tropism for human hepatocytes, increased gene transfer efficiency, and neutralizing antibody evasion. Specifically, AAVhum.8 can better evade neutralizing antisera from multiple species than AAV8. Further, AAVhum.8 displays robust transduction in a human liver xenograft mouse model with expanded tropism for both murine and human hepatocytes. This work supports the hypothesis that critical properties, such as AAV capsid antibody evasion and tropism, can be coevolved by combining rational design and library-based evolution for clinical gene therapy.IMPORTANCE Clinical gene therapy with recombinant AAV vectors has largely relied on natural capsid isolates. There is an unmet need to comprehensively improve AAV tissue tropism, transduction efficiency, and antibody evasion. Such cannot be achieved by utilizing capsid sequence data alone but requires harnessing the 3D structural properties of AAV capsids. Here, we combine rational design and library-based evolution to coevolve multiple, desirable properties onto AAV by harnessing 3D structural information.

Microbubbles for Nucleic Acid Delivery in Liver Using Mild Sonoporation.

Ultrasound-mediated gene delivery is an interesting approach, which could help in increasing gene transfer in deep tissues. Moreover, it allows for performing experiments guided by the image to determine which elements are required. Microbubbles complexed with a eukaryotic expression cassette are excellent agents as they are responsive to ultrasounds and, upon oscillation, can destabilize membranes to enhance gene transfer. Here, we describe the preparation of positively charged microbubbles, plasmid free of antibiotic resistance marker, their combination and the conditions of ultrasound-mediated liver transfection post-systemic administration in mice. This association allowed us to obtain a superior liver gene expression at least over 8months after a single injection.

Prolonging pulse duration in ultrasound-mediated gene delivery lowers acoustic pressure threshold for efficient gene transfer to cells and small animals

While ultrasound-mediated gene delivery (UMGD) has been accomplished using high peak negative pressures (PNPs) of 2 MPa or above, emerging research showed that this may not be a requirement for microbubble (MB) cavitation. Thus, we investigated lower-pressure conditions close to the MB inertial cavitation threshold and focused towards further increasing gene transfer efficiency and reducing associated cell damage. We created a matrix of 21 conditions (n = 3/cond.) to test in HEK293T cells using pulse durations spanning 18 μs–36 ms and PNPs spanning 0.5–2.5 MPa. Longer pulse duration conditions yielded significant increase in transgene expression relative to sham with local maxima between 20 J and 100 J energy curves. A similar set of 17 conditions (n = 4/cond.) was tested in mice using pulse durations spanning 18 μs–22 ms and PNPs spanning 0.5–2.5 MPa. We observed local maxima located between 1 J and 10 J energy curves in treated mice. Of these, several low pressure conditions showed a decrease in ALT and AST levels while maintaining better or comparable expression to our positive control, indicating a clear benefit to allow for effective transfection with minimized tissue damage versus the high-intensity control. Our data indicates that it is possible to eliminate the requirement of high PNPs by prolonging pulse durations for effective UMGD in vitro and in vivo, circumventing the peak power density limitations imposed by piezo-materials used in US transducers. Overall, these results demonstrate the advancement of UMGD technology for achieving efficient gene transfer and potential scalability to larger animal models and human application.

Gene Delivery in Neuro-Oncology.

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults with a dismal prognosis despite aggressive multimodal management thus novel treatments are urgently needed. Gene therapy is a versatile treatment strategy being investigated in multiple cancers including GBM. In gene therapy, a variety of vectors or "carriers" are used to deliver genes designed for different anti-tumoral effects. Gene delivery vehicles and approaches to treatment will be addressed in this review. The most commonly studied vectors are viral based, however, driven by advances in biomedical engineering, mesenchymal and neural stem cells, as well as multiple different types of nanoparticles have been developed to improve tumor tropism and also increase gene transfer into tumor cells. Different genes have been studied including suicide genes, which convert non-toxic prodrug into cytotoxic drug; immunomodulatory genes, which stimulate the immune system; and tumor suppressor genes which repair the defect that allow cells to divide unchecked. Gene therapy may be a promising treatment strategy in neuro-oncology as it is versatile and flexible due to the ability to tailor vectors and genes for specific therapeutic activity. Pre-clinical studies and clinical trials have demonstrated feasibility and safety of gene therapy; however, further studies are required to determine efficacy.

Incremental Innovation of Ex Vivo Hematopoietic Stem Cell Engineering to Expand Clinical Gene Therapy Applications

Transplantation of genetically engineered, autologous hematopoietic stem and progenitor cells (HSPC) is becoming a promising alternative to allogeneic stem cell transplantation for curing genetic diseases, avoiding the risks of graft versus host disease and prolonged immunosuppression. Most clinical gene therapy protocols are based on CD34+ HSPC engineered during >2 days of ex vivo culture. By xenotransplanting mobilized peripheral blood (mPB) CD34+ HSPC, which were lentivirally (LV) marked with different fluorescent proteins according to CD38/CD90 expression levels allowing quantitative assessment of the contribution of CD38/CD90 subpopulations to hematopoietic reconstitution (n=48 NSG mice, 3 experiments), we identified 2 distinct waves of reconstitution: (1) short term repopulation (up to 2 months) mostly driven by CD34+CD38intCD90+/- cells and (2) long-term repopulation driven by CD34+CD38-CD90+ (70%) and CD34+CD38-CD90- cells (30%). Notably, an intermediate wave extending from 2 to 4 months driven by CD34+CD38low cells was selectively eliminated by prolonged ex vivo culture and could be rescued when culture time was reduced to 1 day. We therefore developed a novel LV transduction protocol able to provide curative levels of gene transfer during a single day of ex vivo culture. Stimulating CD34+ cells or CD34+CD38- cells with Prostaglandin E2 (PGE2) increased gene transfer with VSVg-pseudotyped LVs by 1.5-2 fold acting on early steps of transduction, an effect that was further potentiated by the late-acting compound Cyclosporin A. Using large-scale vector preparations for gene therapy of mucopolysaccharidosis type 1, chronic granulomatous disease or beta-thalassemia, we show by in vitro and xenotransplantation assays that a 1-day PGE2 protocol achieved similar transduction efficiencies into BM or MPB HSPC from healthy donors and patients as our 62h benchmark protocol. PGE2 treatment did not result in toxicity or skewed multi-lineage differentiation. However, shortening ex vivo culture increased engraftment levels in the NSG mouse model. To entirely avoid culturing progenitor cells, we explored the feasibility to limit ex vivo manipulation to HSC-enriched CD34+CD38- cells that may be co-transplanted with unmanipulated CD34+ progenitor cells devoid of long-term engraftment potential. This could further improve hematopoietic reconstitution, increase safety by reducing the LV integration load infused into the patient and downscale ex vivo manipulation making the process more efficient and economically sustainable. To this end, we optimized a sequential bead-based, GMP-compatible selection procedure to separate mPB into a CD34+CD38- stem and CD38+ progenitor cell fraction. We reached high purity (87+/-6.6% CD34+) and recovery of CD34+CD38- cells (37.3+/-8.7%), making their isolation clinically viable. Bead-selected CD34+CD38- cells showed higher engraftment potential than equivalent numbers of FACS-sorted cells. Co-infusion of unmanipulated (culture-sensitive) CD38+ supporter cells with genetically-engineered CD34+CD38- cells into NSG mice resulted in rapid engraftment followed by near-complete replacement of untransduced short-term repopulating progenitors by gene-marked HSPC deriving from CD34+CD38- cells after the 3rd month post-transplant. Finally, we explored ex vivo expansion of mPB CD34+CD38- cells with arylhydrocarbon receptor antagonists and/or pyrimido-indole-derivatives. These cells expanded 3-10 fold in a 7-14 d time-window, far less than seen for total CD34+ cells, thereby facilitating culture handling and reducing cost. Unlike CD34+ cells, expanded mPB CD34+CD38- cells largely maintained their SCID-repopulating potential providing proof-of-concept for the expansion of gene-modified HSC. This clinically applicable platform will improve the efficacy, safety and sustainability of ex vivo gene addition and open up new opportunities in the field of gene editing. DisclosuresCiceri:MolMed SpA: Consultancy.

Spatial Control of Gene Expression by Nanocarriers Using Heparin Masking and Ultrasound-Targeted Microbubble Destruction.

We developed a method to spatially control gene expression following nonviral delivery of DNA. This method includes surface-modifying DNA nanocarriers with heparin to inhibit passive gene transfer in both the target and the off-target tissues and using ultrasound-targeted microbubble destruction (UTMD) to selectively activate heparin-inhibited gene transfer at the target site. We observed that the engraftment of heparin onto the surface of cationic liposomes reduced off-target gene expression in the liver, a major site of nanoplex accumulation, by more than 700-fold compared to the nonheparinized PEGylated liposomes. We further observed that tumor-directed UTMD increased gene transfer with heparin-modified nanoplexes by more than 10-fold. This method augmented tumor-to-liver selectivity of gene expression by 4000-fold compared to controls. We conclude that heparinization of DNA nanocarriers in conjunction with localized activation of gene transfer by UTMD may enable greater spatial control over genetic therapy.

356. Ultrasound Power and Pulse Interaction for Effective Gene Delivery in Cells and Animals

While historically ultrasound-mediated gene delivery (UMGD) has been accomplished using high peak negative pressures (PNPs) of 2 MPa or above, emerging research has shown that this is not a requirement for microbubble (MB) cavitation. Thus, we investigated lower-pressure conditions for UMGD, close to the MB inertial cavitation threshold. These conditions were focused towards further increasing gene transfer efficiency and reducing associated cell damage.To test for optimal UMGD conditions in vitro, we created a matrix of 21 conditions (n = 3/cond.) in 293T cells. In an anechoic water bath, we US-treated cells for 60 s in the near field of a 16 mm disc transducer (H158) at 1 MHz with 14 Hz PRF, using pulse durations spanning 18 μs–36 ms and PNPs spanning 450–2370 kPa. Cells were suspended and mixed with pGFP plasmid and MBs in acoustically transparent 1.7 mL TPX tubes during treatment. Using this system, we also performed endpoint studies of MB cavitation using tunable resistive pulse sensing (TRPS) and flow cytometry. Our best 293T conditions yielded up to a 900-fold increase in expression (MFI × % GFP+) relative to sham. We also identified a local maxima with a -3 dB falloff approximately bounded by PNPs 450–1000 kPa and pulses 2–22 ms. Interestingly, in contrast to murine results, the 18 μs, 2480 kPa condition produced no elevated expression relative to sham. Our early mechanistic data showed increased destruction and aggregation of MBs for increasing pulse durations under conditions normalized by energy output. This suggests a different mode of cavitation or higher cavitation efficiency for those conditions.We next tested a similar set of 17 conditions (n = 4/cond) in 8-week old C57BL/6 mice using pulse durations 50 μs–22 ms and PNPs 480–2200 kPa. H158 was applied directly to the liver surface with simultaneous injection of MBs and luciferase plasmid for 60s. These conditions were compared against a short pulse, high-intensity positive control (18 μs, PNP 2540 kPa) which previously yielded the best transgene expression. We found a similar maximum in treated mice, with a -3 dB falloff bounded by PNPs 480-700 kPa. Of these, two conditions (1 & 2 ms, PNP 480 kPa) showed a significant ≈2-fold decrease in ALT and AST (p < 0.005 for all) while maintaining better or comparable expression to our positive control. These results demonstrated a clear benefit of certain low-pressure, millisecond-pulse conditions versus the high-intensity control.Our data indicates that it is possible to eliminate the requirement of high PNPs by prolonging pulse durations for effective UMGD in vitro and in vivo. This study also established a simple, laboratory-feasible methodology for efficient UMGD in cells at low-intensity and millisecond pulse durations. Furthermore, these conditions allow for effective transfection in murine livers with minimized tissue damage. Our ongoing research investigates whether therapeutic effects of our high-intensity controls and lower-intensity, millisecond conditions represent separate cavitation phenomena or a spectrum of a single effect.

317. HH67.2, a Promising Novel Adeno-Associated Virus (AAV) Vector, Shows Improved Muscle Specificity and Infectivity Compared To AAV9

Currently, AAV9 serotype is considered a robust heart and muscle gene delivery vehicle widely used in preclinical studies and in a clinical trial for spinal muscle atrophy (SMA). However, the systemic delivery of AAV9 with large vector doses leads to undesirable effects such as ectopic gene expression in other tissues. These shortcomings may be overcome by using certain strategies such as the development of novel AAV vectors with muscle-specific tropism. Previously, our lab identified a novel muscle specific AAV vector, HH67, by DNA shuffling and in vivo selection. Similar to other evolved tissue-specific vectors, the HH67 vector showed reduced liver tropism, but its transduction efficiency in heart and muscle tissues was still lower than that of AAV9. We set out to introduce site-directed mutations of tyrosine (Y) to phenylalanine (F) on the surface of AAV capsids which has been shown to significantly increase gene transfer efficiency for certain AAV serotypes. In this study, we investigated whether two capsid mutations in HH67, Y445F and Y731F (named HH67.2), would enhance gene delivery to the muscle while maintaining low non-specific transgene expression. We found that the HH67.2 mutant had a significant increase in transgene delivery to the muscle compared to AAV9 after systemic delivery in C57BL/6 mice via tail-vein injection. In the muscle, expression of the LacZ reporter gene (2.5 × 10^5±65413 units/μg HH67.2 vs 7.2 × 10^4±8211 units/μg AAV9; p=0.0049) and the DNA genome copy number (4.50±2.07 VG/cell HH67.2 vs 1.15±0.197 VG/cell AAV9; p=0.0118) both increased 3.5-fold compared to AAV9. Furthermore, liver and other non-specific organs showed up to an 11-fold decrease in HH67.2 vector copy number (11.8±2.5 VG/cell HH67.2 liver vs 126.8±39.6 VG/cell AAV9 liver; p<0.0001). However, HH67.2 and AAV9 had no significant difference in gene expression in the heart. LacZ expression results were confirmed by vector genome copy number via quantitative PCR and X-Gal staining. Finally, we also introduced the same Y-F mutations in three similarly shuffled and in vivo selected AAV capsids such as M41, H43 and H48, but the mutations decreased infectivities of all these capsids. Our results suggest that HH67.2 is a novel AAV vector with enhanced muscle specificity and infectivity compared to AAV9 and could be a promising vehicle to deliver therapeutic genes to skeletal muscle or heart.**This work was part of Carrie Martin and Christina Mayer's research project for the Honors Program at the UNC Eshelman School of Pharmacy.

Intracisternal delivery of NFκB-inducible scAAV2/9 reveals locoregional neuroinflammation induced by systemic kainic acid treatment.

We have previously demonstrated disease-dependent gene delivery in the brain using an AAV vector responding to NFκB activation as a probe for inflammatory responses. This vector, injected focally in the parenchyma prior to a systemic kainic acid (KA) injection mediated inducible transgene expression in the hippocampus but not in the cerebellum, regions, respectively, known to be affected or not by the pathology. However, such a focal approach relies on previous knowledge of the model parameters and does not allow to predict the whole brain response to the disease. Global brain gene delivery would allow to predict the regional distribution of the pathology as well as to deliver therapeutic factors in all affected brain regions. We show that self-complementary AAV2/9 (scAAV2/9) delivery in the adult rat cisterna magna allows a widespread but not homogenous transduction of the brain. Indeed, superficial regions, i.e., cortex, hippocampus, and cerebellum were more efficiently transduced than deeper regions, such as striatum, and substantia nigra. These data suggest that viral particles penetration from the cerebrospinal fluid (CSF) into the brain is a limiting factor. Interestingly, AAV2/9-2YF a rationally designed capsid mutant (affecting surface tyrosines) increased gene transfer efficiency approximately fivefold. Neurons, astrocytes, and oligodendrocytes, but not microglia, were transduced in varying proportions depending on the brain region and the type of capsid. Finally, after a single intracisternal injection of scAAV2/9-2YF using the NFκB-inducible promoter, KA treatment induced transgene expression in the hippocampus and cortex but not in the cerebellum, corresponding to the expression of the CD11b marker of microglial activation. These data support the use of disease-inducible vectors administered in the cisterna magna as a tool to characterize the brain pathology in systemic drug-induced or transgenic disease models. However, further improvements are required to enhance viral particles penetration into the brain.

Improving DNA vaccine performance through vector design.

DNA vaccines are a rapidly deployed next generation vaccination platform for treatment of human and animal disease. DNA delivery devices, such as electroporation and needle free jet injectors, are used to increase gene transfer. This results in higher antigen expression which correlates with improved humoral and cellular immunity in humans and animals. This review highlights recent vector and transgene design innovations that improve DNA vaccine performance. These new vectors improve antigen expression, increase plasmid manufacturing yield and quality in bioreactors, and eliminate antibiotic selection and other potential safety issues. A flowchart for designing synthetic antigen transgenes, combining antigen targeting, codon-optimization and bioinformatics, is presented. Application of improved vectors, of antibiotic free plasmid production, and cost effective manufacturing technologies will be critical to ensure safety, efficacy, and economically viable manufacturing of DNA vaccines currently under development for infectious disease, cancer, autoimmunity, immunotolerance and allergy indications.

Improved gene transfer efficiency of a DNA-lipid-apatite composite layer by controlling the layer molecular composition

Surface-mediated nonviral gene transfer systems using biocompatible apatite-based composite layers have potential use in tissue engineering applications. Herein, we investigated a relatively efficient system based on a DNA-lipid-apatite composite layer (DLp-Ap layer): an apatite (Ap) layer with immobilized DNA and lipid (Lp) complexes (DLp complexes). DLp-Ap layers were fabricated on substrates using supersaturated calcium phosphate coprecipitation solutions supplemented with DLp complexes, and the molecular compositions of the DLp-Ap layers were controlled by varying the net DNA concentrations and Lp/DNA ratios in the coprecipitation solutions. Increases in both the DNA concentration and Lp/DNA ratio in the coprecipitation solution increased the DLp complex content of the resulting DLp-Ap layer. However, a higher DLp complex content did not always provide increased gene transfer efficiency to the CHO-K1 cells, because there was a threshold content of approximately 10μg/cm2. In addition, DLp-Ap layers with similar DLp complex contents exhibited different gene transfer efficiencies, most likely due to the different Lp/DNA ratios in the layers. Notably, the optimized Lp/DNA ratios in the coprecipitation solutions for maximizing the gene transfer efficiency were lower than those of the conventional particle-mediated lipofection systems. These findings will serve as a useful design guide for the preparation of DLp-Ap layers with high gene transfer efficiency.

Interleukin-12 plasmid DNA delivery using l-thyroxine-conjugated polyethylenimine nanocarriers

In this study, l-thyroxine was covalently grafted on 25 kDa branched polyethylenimine (PEI), and the ability of the nano-sized polyplexes for transferring plasmid encoding interleukin-12 (IL-12) gene was evaluated. As there are several problems in systemic administration of recombinant IL-12 protein, local expression of the plasmid encoding IL-12 gene inside the tumor tissue has been considered as an effective alternative approach. The l-thyroxine-conjugated PEI polyplexes were prepared using pUMVC3-hIL12 plasmid, and their transfection activity was determined in HepG2 human liver carcinoma and Neuro2A neuroblastoma cell lines. The polyplexes characterized in terms of DNA condensation ability, particle size, zeta potential, and buffering capacity as well as cytotoxicity and resistance to enzyme digestion. The results revealed that l-thyroxine conjugation of PEI increased gene transfer ability by up to two fold relative to unmodified 25 kDa PEI, the gold standard for non-viral gene delivery, with the highest increase occurring at degrees of conjugation around 10 %. pDNA condensation tests and dynamic light scattering measurements exhibited the ability of PEI conjugates to optimally condense the plasmid DNA into polyplexes in the size range around 200 nm. The modified polymers showed remarkable buffering capacity and protection against enzymatic degradation comparable to that of unmodified PEI. These results suggest that l-thyroxine conjugation of PEI is a simple modification strategy for future investigations aimed at developing a targeting gene vehicle.

Correlation of Length of Linear Oligo(ethanamino) Amides with Gene Transfer and Cytotoxicity

The optimization of synthetic carriers for gene transfer remains a major challenge. Cationic polymers such as polyethylenimine (PEI) often show increasing gene transfer activity with increasing molecular weight, but this favorable effect is accompanied by an undesired increase in cytotoxicity. Moreover, the polydispersity of polymers prevents accurate determination of optimum size. Herein we describe the step-by-step elongation of precise linear oligo(ethanamino) amides by making use of the artificial amino acid succinoyl-tetraethylene pentamine (Stp) for solid-phase-assisted synthesis. This procedure enabled us to identify the optimal oligomer Stp30-W (8.4 kDa) with a length of 30 Stp units, with which effective gene transfer occurs in the absence of cytotoxicity. The transfection efficiency of Stp30-W exceeded that of standard linear PEI (22 kDa) by sixfold; nevertheless, Stp30-W exhibited tenfold lower cytotoxicity. In addition to the lower molecular weight, the succinate spacer between the oligoamine units may also contribute to the favorable biocompatibility. The cytotoxicity of the cationic polymer PEI is a major concern for use as a carrier for gene delivery, so this comparison between linear PEI and the new Stp oligomers is particularly relevant.