Kinetics Of Transgene Expression Research Articles

Immune responses, therapeutic anti-tumor effects, and tolerability upon therapeutic HPV16/18 E6/E7 DNA vaccination via needle-free biojector.

Respectively, HPV16 and HPV18 cause 50% and 20% of cervical cancer cases globally. Viral proteins E6 and E7 are obligate drivers of oncogenic transformation. We recently developed a candidate therapeutic DNA vaccine, pBI-11, that targets HPV16 and HPV18 E6 and E7. Single-site intramuscular delivery of pBI-11 via a needle elicited therapeutic anti-tumor effects in mice and is now being tested in high-risk human papillomavirus+ head and neck cancer patients (NCT05799144). Needle-free biojectors such as the Tropis device show promise due to ease of administration, high patient acceptability, and the possibility of improved delivery. For example, vaccination of patients with the ZyCoV-D DNA vaccine using the Tropis device is effective against COVID19, well tolerated, and licensed. Here we show that split-dose, multi-site administration and intradermal delivery via the Tropis biojector increase the delivery of pBI-11 DNA vaccine, enhance HPV antigen-specific CD8+ T-cell responses, and improve anti-tumor therapeutic effects, suggesting its translational potential to treat HPV16/18 infection and disease.

Kinetics and durability of transgene expression after intrastriatal injection of AAV9 vectors.

Understanding the kinetics and durability of AAV-mediated transgene expression in the brain is essential for conducting basic neuroscience studies as well as for developing gene therapy approaches for CNS diseases. Here, we characterize and compare the temporal profile of transgene expression after bilateral injections into the mouse striatum of rAAV9 encoding GFP under the control of either a ubiquitous promoter (CAG), or the neuron-specific human synapsin (hSyn) and CamKII promoters. GFP protein expression with the CAG promoter was highest at 3 weeks, and then decreased to stable levels at 3 and 6 months. Surprisingly, GFP mRNA levels continued to increase from 3 weeks to 3 months, despite GFP protein expression decreasing during this time. GFP protein expression with hSyn increased more slowly, reaching a maximum at 3 months, which was equivalent to protein expression levels from CAG at that time point. Importantly, transgene expression driven by the hSyn promoter at 6 months was not silenced as previously reported, and GFP mRNA was continuing to rise even at the final 6-month time point. Thus, hSyn as a promoter for transgene expression demonstrates long-term durability but may require more time after vector administration to achieve steady-state levels. Because CAG had the highest GFP protein expression in our comparison, which was at 3 weeks post administration, the early kinetics of transgene expression from CAG was examined (1, 2, 5, and 10 days after injection). This analysis showed that GFP protein expression and GFP mRNA increased during the first 3 weeks after administration. Interestingly, vector DNA rapidly decreased 10-fold over the first 3 weeks following injection as it assembled into stable circular episomes and concatemers. Surprisingly, the processing of vector genomes into circular episomes and concatemers was continually dynamic up to 3 months after injection. These results provide novel insight into the dynamic processing of vector genomes and promoter-specific temporal patterns of transgene expression in the brain.

S233: THERAPEUTIC GENE EDITING OF T CELLS CORRECTS CTLA4 INSUFFICIENCY.

Background: Heterozygous mutations in CTLA4 result in an inborn error of immunity (IEI) (also known as primary immunodeficiency) with a severe clinical phenotype. Autologous T cell gene therapy may offer a cure without the immunological complications of allogeneic stem cell transplantation. The mutational landscape and requirement for tight regulation of CTLA4 make viral gene addition approaches unappealing. Gene editing strategies permit alteration of CTLA4 while retaining the endogenous gene control machinery. Aims: We set out to devise a CRISPR/Cas9/AAV6 gene editing strategy to correct CTLA4 insufficiency in T cells. Methods: We designed several homology directed repair (HDR) editing strategies that would correct the genetic defect. We first assessed correction of an individual point mutation. We then evaluated several universal strategies that enable correction of most disease-causing mutations with a single edit; the first that inserts the CTLA4 cDNA in exon 1, and a second that inserts the CTLA4 cDNA at the 3’end of the first intron of CTLA4. All AAV6 HDR donor templates included a GFP reporter gene to enable easy identification of the edited cells. Results: Superior editing efficiencies were obtained with the intronic approach compared to the other editing strategies and this strategy was then further evaluated. CTLA4 function and expression kinetics were assessed following editing using flow cytometry-based assays. Functional studies using CTLA4 transendocytosis (TE) assays, demonstrated restoration of CD80 and CD86 internalization in the edited CD4+ T cells. Following gene editing, transgene expression kinetics were comparable to healthy control CD4+ T cells. Gene editing of T cells isolated from patients with CTLA4 insufficiency restored CTLA4 expression and rescued transendocytosis of CD80 and CD86 in vitro. Using a similar approach, gene corrected T cells from CTLA4-/- mice engrafted in immunodeficient mice at clinically relevant frequencies and tail vein bleeds were performed 1, 3 and 4 weeks post adoptive transfer. In the mice which received the GFP+ edited cells, a stable population of GFP+ cells was detectable at all timepoints demonstrating in vivo persistence as well as genetic stability. All mice were sacrificed 4 weeks after cell transfer. To assess lymphoproliferation, the cellularity of peripheral lymph nodes and spleen weight were analyzed. Spleen and lymph node size, lymph node cell counts, and spleen weight were all significantly lower in mice which received the edited cells (n=5) compared to non-edited controls (n=5) and there was no significant difference in these parameters between mice who had received edited CTLA4-/- T cells and those who received WT T cells (n=4). Analysis of lymph node and spleen cells confirmed persistence of CTLA4 expression in the edited (GFP+) cells and revealed that a higher proportion of Treg than Tconv had been successfully edited. Summary/Conclusion: Together these data demonstrated that CTLA4 edited T cells survived in vivo, expressed CTLA4 and were able to control the clinical phenotype of CTLA4 insufficiency, providing a powerful proof-of-principle of our T cell GT approach. Our data provide proof-of-concept that gene editing can restore CTLA4 function in T cells demonstrating the potential of this approach to treat CTLA4 insufficiency. A similar approach could be used in other IEIs that are caused by multiple heterozygous mutations.

Inclusion of a dual signal sequence enhances the immunogenicity of a novel viral vectored vaccine against the capsular group B meningococcus

BackgroundDisease caused by the capsular group B meningococcus (MenB) is the leading cause of infectious death in UK infants. A novel adenovirus-based vaccine encoding the MenB factor H binding protein (fHbp) with an N-terminal dual signal sequence induces high titres of protective antibody after a single dose in mice. A panel of N-terminal signal sequence variants were created to assess the contribution of components of this sequence to transgene expression kinetics of the encoded antigen from mammalian cells and the resultant effect on immunogenicity of fHbp.ResultsThe full-length signal sequence (FL SS) resulted in superior early antigen expression compared with the panel of variants, as measured by flow cytometry and confocal imaging, and supported higher bactericidal antibody levels against the expressed antigen in mouse sera < 6 weeks post-immunisation than the licensed four component MenB vaccine. The FL SS also significantly increased antigen-specific T cell responses against other adenovirus-encoded bacterial antigens in mice.ConclusionsThese findings demonstrate that the FL SS enhances immunogenicity of the encoded antigen, supporting its inclusion in other viral vectored bacterial antigen transgenes.

MicroRNA-sensitive oncolytic measles virus for chemovirotherapy of pancreatic cancer

Advanced pancreatic cancer is characterized by few treatment options and poor outcomes. Oncolytic virotherapy and chemotherapy involve complementary pharmacodynamics and could synergize to improve therapeutic efficacy. Likewise, multimodality treatment may cause additional toxicity, and new agents have to be safe. Balancing both aims, we generated an oncolytic measles virus for 5-fluorouracil-based chemovirotherapy of pancreatic cancer with enhanced tumor specificity through microRNA-regulated vector tropism. The resulting vector encodes a bacterial prodrug convertase, cytosine deaminase-uracil phosphoribosyl transferase, and carries synthetic miR-148a target sites in the viral F gene. Combination of the armed and targeted virus with 5-fluorocytosine, a prodrug of 5-fluorouracil, resulted in cytotoxicity toward both infected and bystander pancreatic cancer cells. In pancreatic cancer xenografts, a single intratumoral injection of the virus induced robust in vivo expression of prodrug convertase. Based on intratumoral transgene expression kinetics, we devised a chemovirotherapy regimen to assess treatment efficacy. Concerted multimodality treatment with intratumoral virus and systemic prodrug administration delayed tumor growth and prolonged survival of xenograft-bearing mice. Our results demonstrate that 5-fluorouracil-based chemovirotherapy with microRNA-sensitive measles virus is an effective strategy against pancreatic cancer at a favorable therapeutic index that warrants future clinical translation.

Improvement of HSV-1 based amplicon vectors for a safe and long-lasting gene therapy in non-replicating cells

A key factor for developing gene therapy strategies for neurological disorders is the availability of suitable vectors. Currently, the most advanced are adeno-associated vectors that, while being safe and ensuring long-lasting transgene expression, have a very limited cargo capacity. In contrast, herpes simplex virus-based amplicon vectors can host huge amounts of foreign DNA, but concerns exist about their safety and ability to express transgenes long-term. We aimed at modulating and prolonging amplicon-induced transgene expression kinetics in vivo using different promoters and preventing transgene silencing. To pursue the latter, we deleted bacterial DNA sequences derived from vector construction and shielded the transgene cassette using AT-rich and insulator-like sequences (SAm technology). We employed luciferase and GFP as reporter genes. To determine transgene expression kinetics, we injected vectors in the hippocampus of mice that were longitudinally scanned for bioluminescence for 6 months. To evaluate safety, we analyzed multiple markers of damage and performed patch clamp electrophysiology experiments. All vectors proved safe, and we managed to modulate the duration of transgene expression, up to obtaining a stable, long-lasting expression using the SAm technology. Therefore, these amplicon vectors represent a flexible, efficient, and safe tool for gene delivery in the brain.

Control of Microbial Opsin Expression in Stem Cell Derived Cones for Improved Outcomes in Cell Therapy.

Human-induced pluripotent stem cell (hiPSC) derived organoids have become increasingly used systems allowing 3D-modeling of human organ development, and disease. They are also a reliable source of cells for transplantation in cell therapy and an excellent model to validate gene therapies. To make full use of these systems, a toolkit of genetic modification techniques is necessary to control their activity in line with the downstream application. We have previously described adeno-associated viruse (AAV) vectors for efficient targeting of cells within human retinal organoids. Here, we describe biological restriction and enhanced gene expression in cone cells of such organoids thanks to the use of a 1.7-kb L-opsin promoter. We illustrate the usefulness of implementing such a promoter to enhance the expression of the red-shifted opsin Jaws in fusion with a fluorescent reporter gene, enabling cell sorting to enrich the desired cell population. Increased Jaws expression after transplantation improved light responses promising better therapeutic outcomes in a cell therapy setting. Our results point to the importance of promoter activity in restricting, improving, and controlling the kinetics of transgene expression during the maturation of hiPSC retinal derivatives. Differentiation requires mechanisms to initiate specific transcriptional changes and to reinforce those changes when mature cell states are reached. By employing a cell-type-specific promoter we put transgene expression under the new transcriptional program of mature cells.

Microfluidic Electroporation Coupling Pulses of Nanoseconds and Milliseconds to Facilitate Rapid Uptake and Enhanced Expression of DNA in Cell Therapy

Standard electroporation with pulses in milliseconds has been used as an effective tool to deliver drugs or genetic probes into cells, while irreversible electroporation with nanosecond pulses is explored to alter intracellular activities for pulse-induced apoptosis. A combination treatment, long nanosecond pulses followed by standard millisecond pulses, is adopted in this work to help facilitate DNA plasmids to cross both cell plasma membrane and nuclear membrane quickly to promote the transgene expression level and kinetics in both adherent and suspension cells. Nanosecond pulses with 400–800 ns duration are found effective on disrupting nuclear membrane to advance nuclear delivery of plasmid DNA. The additional microfluidic operation further helps suppress the negative impacts such as Joule heating and gas bubble evolution from common nanosecond pulse treatment that lead to high toxicity and/or ineffective transfection. Having appropriate order and little delay between the two types of treatment with different pulse duration is critical to guarantee the effectiveness: 2 folds or higher transfection efficiency enhancement and rapid transgene expression kinetics of GFP plasmids at no compromise of cell viability. The implementation of this new electroporation approach may benefit many biology studies and clinical practice that needs efficient delivery of exogenous probes.

Engineered Mesenchymal Stromal Cell Therapy during Pig Ex Vivo Lung Perfusion and Transplant

Purpose Ex vivo lung perfusion (EVLP) enables novel therapeutic approaches to repair damaged lungs for transplantation. We investigated the concept and feasibility of elective cell therapy for donor lungs. Dosing, efficacy and transgene expression kinetics of engineered mesenchymal stromal cell therapy were examined in pig EVLP and transplantation. Methods Human umbilical cord perivascular mesenchymal stromal/stem cells were transduced with an adenoviral vector encoding human IL-10 (MSC-hIL-10 cells), cryopreserved and recovered 1h before use. Pig lungs with 4 to 24-hour cold ischemia (n=5) were connected to EVLP for 6 (n=3) or 12 hours (n=2) and MSC-hIL-10 cells (40-150 × 106) were administered through the pulmonary artery after 1h of EVLP. Left single lung transplantation was performed after EVLP and the grafts were monitored up to 4h (n=4) and 3d (n=1) after reperfusion. Human IL-10 levels were determined by ELISA and fluorescence in situ hybridization with human-specific ALU probes was used to track MSC-hIL-10 cells. Results Supra-therapeutic levels of hIL-10 were present in EVLP perfusate within minutes after MSC-hIL10 cell administration and the levels consistently increased during EVLP (Figure 1A). Recipient plasma hIL-10 levels were elevated 1h after lung transplantation, remained high for 4h and gradually decreased to low levels by 3d after transplantation (Figure 1C). Elevated tissue and bronchoalveolar lavage hIL-10 levels were observed in the transplanted lung and remained low in the recipient native lung. MSC-hIL-10 cells were detected in the donor lung during EVLP and after transplantation (Figure 1B). Conclusion Engineered mesenchymal stromal cell therapy during EVLP is a novel and practical “off-the-shelf” strategy to achieve rapid transient therapeutic levels of soluble cytokines such as IL-10 for pre-transplant lung repair and protection. Ongoing studies will determine the therapeutic effect and longer-term fate of MSC-hIL-10 cells delivered during EVLP.

Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

BackgroundMessenger RNA (mRNA) has gained remarkable attention as an alternative to DNA-based therapies in biomedical research. A variety of biodegradable nanoparticles (NPs) has been developed including lipid-based and polymer-based systems for mRNA delivery. However, both systems still lack in achieving an efficient transfection rate and a detailed understanding of the mRNA transgene expression kinetics. Therefore, quantitative analysis of the time-dependent translation behavior would provide a better understanding of mRNA’s transient nature and further aid the enhancement of appropriate carriers with the perspective to generate future precision nanomedicines with quick response to treat various diseases.ResultsA lipid–polymer hybrid system complexed with mRNA was evaluated regarding its efficiency to transfect dendritic cells (DCs) by simultaneous live cell video imaging of both particle uptake and reporter gene expression. We prepared and optimized NPs consisting of poly (lactid-co-glycolid) (PLGA) coated with the cationic lipid 1, 2-di-O-octadecenyl-3-trimethylammonium propane abbreviated as LPNs. An earlier developed polymer-based delivery system (chitosan-PLGA NPs) served for comparison. Both NPs types were complexed with mRNA-mCherry at various ratios. While cellular uptake and toxicity of either NPs was comparable, LPNs showed a significantly higher transfection efficiency of ~ 80% while chitosan-PLGA NPs revealed only ~ 5%. Further kinetic analysis elicited a start of protein translation after 1 h, with a maximum after 4 h and drop of transgene expression after 48 h post-transfection, in agreement with the transient nature of mRNA.ConclusionsCharge-mediated complexation of mRNA to NPs enables efficient and fast cellular delivery and subsequent protein translation. While cellular uptake of both NP types was comparable, mRNA transgene expression was superior to polymer-based NPs when delivered by lipid–polymer NPs.

Deciphering the impact of parameters influencing transgene expression kinetics after repeated cell transduction with integration-deficient retroviral vectors.

Lentiviral and gammaretroviral vectors are state-of-the-art tools for transgene expression within target cells. The integration of these vectors can be deliberately suppressed to derive a transient gene expression system based on extrachromosomal circular episomes with intact coding regions. These episomes can be used to deliver DNA templates and to express RNA or protein. Importantly, transient gene transfer avoids the genotoxic side effects of integrating vectors. Restricting their applicability, episomes are rapidly lost upon dilution in dividing target cells. Addressing this limitation, we could establish comparably stable percentages of transgene-positive cells over prolonged time periods in proliferating cells by repeated transductions. Flow cytometry was applied for kinetic analyses to decipher the impact of individual parameters on the kinetics of fluoroprotein expression after episomal retransduction and to visualize sequential and simultaneous transfer of heterologous fluoroproteins. Expression windows could be exactly timed by the number of transduction steps. The kinetics of signal loss was affected by the cell proliferation rate. The transfer of genes encoding fluoroproteins with different half-lives revealed a major impact of protein stability on temporal signal distribution and accumulation, determining optimal retransduction intervals. In addition, sequential transductions proved broad applicability in different cell types and using different envelope pseudotypes without receptor overload. Stable percentages of cells coexpressing multiple transgenes could be generated upon repeated coadministration of different episomal vectors. Alternatively, defined patterns of transgene expression could be recapitulated by sequential transductions. Altogether, we established a methodology to control and adjust a temporally defined window of transgene expression using retroviral episomal vectors. Combined with the highly efficient cell entry of these vectors while avoiding integration, the developed technology is of great significance for a broad panel of applications, including transcription-factor-based induced cell fate conversion and controlled transfer of genetically encoded RNA- or protein-based drugs.

Kinetics of Adeno-Associated Virus Serotype 2 (AAV2) and AAV8 Capsid Antigen PresentationIn VivoAre Identical

Adeno-associated viral (AAV) vectors 2 and 8 have been used in clinical trials for patients with hemophilia, and data suggest that the capsid-specific CD8⁺ T cell response has had a negative impact on therapeutic success. To date the pattern of capsid cross-presentation from AAV2 and AAV8 transduction in vivo has not been elucidated. Previously, we have demonstrated that an engineered AAV2 virus carrying the immune-dominant SIINFEKL peptide in the capsid backbone was indistinguishable from wild type with respect to titer, tropism, and the ability to induce capsid-specific CD8⁺ T cell responses in vivo. In this study, we used the same strategy to engineer an AAV8 vector and demonstrated that antigen from SIINFEKL peptide-integrated AAV8 capsid was effectively presented via either plasmid transfection or AAV8 transduction in vitro. The tissue tropism and transgene expression kinetics of the engineered AAV8 vector in vivo were identical to that of wild-type AAV8. Animal studies show that capsid antigen presentation from AAV transduction was dose dependent, and more importantly, the proliferation of capsid-specific CD8⁺ T cells had similar kinetics (detectable before 30 days and undetectable after 40 days) for both AAV2 and AAV8 vectors. Elucidation of the kinetics of capsid antigen presentation from AAV transduction by various serotypes provides new insight into the potential impact CD8⁺ T cells can have during clinical trials and may help with rational design of effective strategies to prevent capsid-specific CD8⁺ T cell-mediated elimination of AAV-transduced target cells.

Direct Head-To-Head Comparison of Cationic Liposome-Mediated Gene Delivery to Mesenchymal Stem/Stromal Cells of Different Human Sources: A Comprehensive Study

Nonviral gene delivery to human mesenchymal stem/stromal cells (MSC) can be considered a very promising strategy to improve their intrinsic features, amplifying the therapeutic potential of these cells for clinical applications. In this work, we performed a comprehensive comparison of liposome-mediated gene transfer efficiencies to MSC derived from different human sources-bone marrow (BM MSC), adipose tissue-derived cells (ASC), and umbilical cord matrix (UCM MSC). The results obtained using a green fluorescent protein (GFP)-encoding plasmid indicated that MSC isolated from BM and UCM are more amenable to genetic modification when compared to ASC as they exhibited superior levels of viable, GFP(+) cells 48 hr post-transfection, 58 ± 7.1% and 54 ± 3.8%, respectively, versus 33 ± 4.7%. For all cell sources, high cell recoveries (≈50%) and viabilities (>85%) were achieved, and the transgene expression was maintained for 10 days. Levels of plasmid DNA uptake, as well as kinetics of transgene expression and cellular division, were also determined. Importantly, modified cells were found to retain their characteristic immunophenotypic profile and multilineage differentiation capacity. By using the lipofection protocol optimized herein, we were able to maximize transfection efficiencies to human MSC (maximum of 74% total GFP(+) cells) and show that lipofection is a promising transfection strategy for MSC genetic modification, especially when a transient expression of a therapeutic gene is required. Importantly, we also clearly demonstrated that intrinsic features of MSC from different sources should be taken into consideration when developing and optimizing strategies for MSC engineering with a therapeutic gene.

Transfection efficiency and transgene expression kinetics of mRNA delivered in naked and nanoparticle format

Transfection efficiencies and transgene expression kinetics of messenger RNA (mRNA), an emerging class of nucleic acid-based therapeutics, have been poorly characterized. In this study, we evaluated transfection efficiencies of mRNA delivered in naked and nanoparticle format in vitro and in vivo using GFP and luciferase as reporters. While mRNA nanoparticles transfect primary human and mouse dendritic cells (DCs) efficiently in vitro, naked mRNA could not produce any detectable gene product. The protein expression of nanoparticle-mediated transfection in vitro peaks rapidly within 5–7h and decays in a biphasic manner. In vivo, naked mRNA is more efficient than mRNA nanoparticles when administered subcutaneously. In contrast, mRNA nanoparticle performs better when administered intranasally and intravenously. Gene expression is most transient when delivered intravenously in nanoparticle format with an apparent half-life of 1.4h and lasts less than 24h, and most sustained when delivered in the naked format subcutaneously at the base of tail with an apparent half-life of 18h and persists for at least 6days. Notably, exponential decreases in protein expression are consistently observed post-delivery of mRNA in vivo regardless of the mode of delivery (naked or nanoparticle) or the site of administration. This study elucidates the performance of mRNA transfection and suggests a niche for mRNA therapeutics when predictable in vivo transgene expression kinetics is imperative.

Design and characterization of microporous hyaluronic acid hydrogels for in vitro gene transfer to mMSCs

The effective and sustained delivery of DNA locally could increase the applicability of gene therapy in tissue regeneration and therapeutic angiogenesis. One promising approach is to use porous hydrogel scaffolds to encapsulate and deliver nucleotides in the form of nanoparticles to the affected sites. We have designed and characterized microporous (μ-pore) hyaluronic acid hydrogels which allow for effective cell seeding in vitro post-scaffold fabrication and allow for cell spreading and proliferation without requiring high levels of degradation. These factors, coupled with high loading efficiency of DNA polyplexes using a previously developed caged nanoparticle encapsulation (CnE) technique, then allowed for long-term sustained transfection and transgene expression of incorporated mMSCs. In this study, we examined the effect of pore size on gene transfer efficiency and the kinetics of transgene expression. For all investigated pore sizes (30, 60, and 100μm), encapsulated DNA polyplexes were released steadily, starting by day 4 for up to 10days. Likewise, transgene expression was sustained over this period, although significant differences between different pore sizes were not observed. Cell viability was also shown to remain high over time, even in the presence of high concentrations of DNA polyplexes. The knowledge acquired through this in vitro model can be utilized to design and better predict scaffold-mediated gene delivery for local gene therapy in an in vivo model where host cells infiltrate the scaffold over time.

Ex Vivo Adenoviral Vector Gene Delivery Results in Decreased Vector-associated Inflammation Pre- and Post–lung Transplantation in the Pig

Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1β levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function.

Oncolytic Herpes simplex virus expressing yeast cytosine deaminase: relationship between viral replication, transgene expression, prodrug bioactivation

Yeast cytosine deaminase (yCD) is a well-characterized prodrug/enzyme system that converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), and has been combined with oncolytic viruses. However, in vivo studies of the interactions between 5-FC bioactivation and viral replication have not been previously reported, nor have the kinetics of transgene expression and the pharmacokinetics of 5-FC and 5-FU. We constructed a replication-conditional HSV-1 expressing yCD and examined cytotoxicity when 5-FC was initiated at different times after viral infection, and observed that earlier 5-FC administration led to greater cytotoxicity than later 5-FC administration in vitro and in vivo. Twelve days of 5-FC administration was superior to 6 days in animal models, but dosing beyond 12 days did not further enhance efficacy. Consistent with the dosing schedule results, both viral genomic DNA copy number and viral titers were observed to peak on Day 3 after viral injection and gradually decrease thereafter. The virus is replication-conditional and was detected in tumors for as long as 2 weeks after viral injection. The maximum relative extent of yCD conversion of 5-FC to 5-FU in tumors was observed on Day 6 after viral injection and it decreased progressively thereafter. The observation that 5-FU generation within tumors did not lead to appreciable levels of systemic 5-FU (<10 ng/ml) is important and has not been previously reported. The approaches used in these studies of the relationship between the viral replication kinetics, transgene expression, prodrug administration and anti-tumor efficacy are useful in the design of clinical trials of armed, oncolytic viruses.

Native Molecular State of Adeno-Associated Viral Vectors Revealed by Single-Molecule Sequencing

The single-stranded genome of adeno-associated viral (AAV) vectors is one of the key factors leading to slow-rising but long-term transgene expression kinetics. Previous molecular studies have established what is now considered a textbook molecular model of AAV genomes with two copies of inverted tandem repeats at either end. In this study, we profiled hundreds of thousands of individual molecules of AAV vector DNA directly isolated from capsids, using single-molecule sequencing (SMS), which avoids any intermediary steps such as plasmid cloning. The sequence profile at 3' ends of both the regular and oversized vector did show the presence of an inverted terminal repeat (ITR), which provided direct confirmation that AAV vector packaging initiates from its 3' end. Furthermore, the vector 5'-terminus profile showed inconsistent termination for oversized vectors. Such incomplete vectors would not be expected to undergo canonical synthesis of the second strand of their genomic DNA and thus could function only via annealing of complementary strands of DNA. Furthermore, low levels of contaminating plasmid DNA were also detected. SMS may become a valuable tool during the development phase of vectors that are candidates for clinical use and for facilitating/accelerating studies on vector biology.

Transfection efficiency and intracellular fate of polycation liposomes combined with protamine

Endosomal escape and nuclear entry are the two main barriers for successful non-viral gene delivery. To overcome these barriers, polyethylenimine (PEI) with a molecular weight of 800, conjugated to cholesterol (PEI 800-Chol) was synthesized to prepare polycation liposomes (PCLs). The effect of cationic polymers on transfection was investigated by pre-condensing DNA with these before using PCLs. The complexes of PCLs and protamine/DNA nanoparticles (PLPD) were introduced as efficient gene transfer vectors, and displayed obviously higher transfection efficiency (approximately 39-fold) than PCLs/DNA complexes. Kinetics of transgene expression indicated PLPD complexes could be maintained at a relatively high level over 72 h. The order of protamine addition affected the transfection of PLPD complexes. Pre-mixed and post-mixed PLPD complexes improved transfection, although the former was preferred. Distribution of FAM-labeled oligonucleotides (FAM-ODN) in cells mediated by PCLs were throughout the whole cell, while most FAM-ODN were nuclear when transfected with PLPD. These results suggest that the protonation of PEI and membrane destabilization of 1, 2-Dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE) increases the endosomal escape ability of vectors. The addition of protamine, containing nuclear localization signals, improved nuclear entry of DNA. The internalization pathways for PCLs involved multiple processes and were possibly dependent on cell lines.

Soluble TNF-α receptor secretion from healthy or dystrophic mice after AAV6-mediated muscle gene transfer

Muscle is an attractive target because it is easily accessible; it also offers a permissive environment for adeno-associated virus (AAV)-mediated gene transfer and has an abundant blood vascular supply providing an efficient transport system for the secretion of proteins. However, gene therapy of dystrophic muscle may be more difficult than that of healthy tissue because of degenerative-regenerative processes, and also because of the inflammatory context. In this study we followed the expression levels of secreted inhibitors of the proinflammatory tumor necrosis factor (TNF) cytokine after intramuscular (i.m.) injection of AAV6 into dystrophic mdx and healthy C57BL/10 mice. We used two chimeric proteins, namely, the human or murine TNF-soluble receptor I fused with the murine heavy immunoglobulin chain. We conducted an AAV6 dose-response study and determined the kinetics of transgene expression. In addition, we followed the antibody response against the transgenes and studied their expression pattern in the muscle. Our results show that transduction efficiency is reduced in dystrophic muscles as compared with healthy ones. Furthermore, we found that the immune response against the secreted protein is stronger in mdx mice. Together, our results underscore that the pathological state of the muscle has to be taken into consideration when designing gene therapy approaches.