Long-term Transgene Expression Research Articles

Lentiviral Transduction of Face and Limb Flaps

Ex vivo introduction of an immunomodulatory transgene into a face or hand allograft may improve the risk-to-benefit ratio of vascularized composite allografts. Abrogation of the immunogenicity of the skin component of a face or hand allograft may decrease alloreactivity and permit the induction of immunologic tolerance. Proof-of-principle demonstrations of transduction of composite tissue have been established using adenoviral vectors, producing transient gene expression. The authors hypothesized that transduction, integration, and long-term expression of transgenes in a vascularized composite allograft could be achieved using lentiviral vectors. Ex vivo transduction of heterogeneous primary rat cell lines representative of a composite tissue flap's cellular architecture was performed using a luc-enhanced green fluorescent protein (eGFP) human immunodeficiency virus-1-based lentiviral vector. Ex vivo injections of rat superficial inferior epigastric artery flaps with the viral vector were performed intraarterially, intramuscularly, and intradermally. Quantifiable reporter expression by flow cytometry (fluorescence-activated cell sorting) analysis and in vitro bioluminescence was observed. The luc-eGFP vector exhibited broad tropism and allowed transgene expression in relevant cell lines and throughout the flaps. Ex vivo intradermal transfection resulted in genomic integration and long-term constitutive gene expression (>150 days). Similarly, efficient intradermal transfection of face and hand flaps in a rat model corroborated this approach. Ex vivo intravascular perfusion of the vector proved inferior to intradermal injection. Intradermal delivery of the transgenes proved superior to intravascular perfusion. Optimization of this gene-delivery approach may allow long-term, constitutive expression of immunomodulatory proteins in face and hand allografts. Future goals include replacement of the luciferase and eGFP reporter genes with key immunomodulatory proteins.

Lentiviral vectors carrying enhancer elements of Hb9 promoter drive selective transgene expression in mouse spinal cord motor neurons

Recombinant lentiviral vectors (rLVs) have emerged as versatile tools for gene delivery applications due to a number of favorable features, such as the possibility to maintain long-term transgene expression, the flexibility in the design of the expression cassettes and recent improvements in their biosafety profile. Since rLVs are able to infect multiple cell types including post-mitotic cells such as neurons and skeletal muscle cells, several studies have been exploring their application for the study and cure of neurodegenerative diseases. In particular, the introduction of rLVs carrying cell-type specific promoters could restrict the transgene expression either to neuronal or glial cells, thus helping to better dissect in vivo the role played by these cell populations in several neurodegenerative processes. In this study we developed rLVs carrying motor neuron specific regulatory sequences derived from the promoter of homeobox gene Hb9, and demonstrated that these constructs can represent a suitable platform for selective gene-targeting of murine spinal cord motor neurons, in vivo. This tool could be instrumental in the dissection of the molecular mechanisms involved in the selective degeneration of motor neurons occurring in Motor Neuron Diseases.

Activation of the ERK1/2 pathway by the CaMEK gene via adeno-associated virus serotype 9 in cardiomyocytes

Extracellular signal-regulated kinase (ERK1/2) is one of the mitogen-activated protein kinases, key components of the reperfusion injury salvage kinase pathway, which plays an important role in protecting the myocardium from lethal ischemia-reperfusion injury. Constitutive activation of the mitogen-activated protein kinase kinase 1 (CaMEK) can promote ERK1/2 expression, which is thereby expected to exert protective action on the heart against ischemia-reperfusion injury. The adeno-associated virus serotype 9 vector (AVV9) is a novel tool for gene therapies targeting human diseases owing to its nonpathogenic capability for transducing nondividing cells and its long-term transgene expression. We used a recombinant AAV9 vector to deliver the CaMEK gene into cardiomyocytes and assessed whether AAV9 vector-mediated CaMEK gene transfection could enhance the long-term expression and activity of ERK1/2. Our observations suggest that AAV9-mediated gene expression is preferentially restricted to cardiomyocytes and that mediated CaMEK gene transfection enhanced the expression of ERK1/2 phosphorylation and consequently upregulated the expression of downstream components of ERK1/2 and its transcription factors.

Evaluation of Long-Term Gene Expression in Mouse Liver Using PhiC31 Integrase and Hydrodynamic Injection

PhiC31 integrase has the potential to achieve long-term transgene expression because of site-specific and unidirectional recombination. In this study, plasmid DNA (pDNA) encoding Gaussia luciferase (Gluc) cDNA was constructed and the optimal condition for long-term gene expression using phiC31 integrase in mouse liver after hydrodynamic injection was investigated. Gluc is secreted and thus allows quantification of its expression level in blood and easy determination of the expression time-course. Mice were co-transfected with 25 µg donor pDNA (pORF-Gluc/attB) and different amounts of helper pDNA (pCMV-int; from 1 to 50 µg). Serum Gluc expression was evaluated during 120 d. The most highly sustained gene expression was obtained when 10 µg of helper pDNA was co-transfected in ICR and C57BL/6 mice. However, the Gluc expression in C57BL/6 mice was slightly lower and less durable than that in ICR mice. Little hepatic damage caused by phiC31 integrase was observed during 120 d in ICR and C57BL/6 mice.

Identification of adeno-associated viral vectors suitable for intestinal gene delivery and modulation of experimental colitis

Effective gene transfer with sustained gene expression is an important adjunct to the study of intestinal inflammation and future therapy in inflammatory bowel disease. Recombinant adeno-associated virus (AAV) vectors are ideal for gene transfer and long-term transgene expression. The purpose of our study was to identify optimal AAV pseudotypes for transduction of the epithelium in the small intestine and colon, which could be used for studies in experimental colitis. The tropism and transduction efficiencies of AAV pseudotypes 1-10 were examined in murine small intestine and colon 8 wk after administration by real-time PCR and immunohistochemistry. The clinical and histopathological effects of IL-10-mediated intestinal transduction delivered by AAVrh10 were examined in the murine IL-10⁻/⁻ enterocolitis model. Serum IL-10 levels and IL-10 expression were followed by ELISA and real-time PCR, respectively. AAV pseudotypes 4, 7, 8, 9, and 10 demonstrated optimal intestinal transduction. Transgene expression was sustained 8 wk after administration and was frequently observed in enteroendocrine cells. Long-term IL-10 gene expression and serum IL-10 levels were observed following AAV transduction in an IL-10-/- model of enterocolitis. Animals treated with AAVrh10-IL-10 had lower disease activity index scores, higher colon weight-to-length ratios, and lower microscopic inflammation scores. This study identifies novel AAV pseudotypes with small intestine and colon tropism and sustained transgene expression capable of modulating mucosal inflammation in a murine model of enterocolitis.

The Ubiquitous Chromatin Opening Element (UCOE) Enhances Lentiviral Cytidine Deaminase (CDD) Expression and Drug Resistance During Hematopoietic Differentiation of Murine Induced Pluripotent Stem Cells (iPSCs),

Abstract 4179 Introduction:Transfer of drug resistance genes to the hematopoietic system has been advocated for myeloprotection during anti-cancer chemotherapy, however, for malignancies manifestated in blood or bone marrow compartments this strategy carries the risk of inadvertent transduction of tumor cells. Hematopoietic differentiation of induced pluripotent stem cells (iPSCs) has the potential to overcome this problem, and we here have investigated this concept in the context of Cytidine Deaminase (CDD)-mediated myeloprotection. However, epigenetic silencing of transgenic promoter/enhancer elements during differentiation is a major drawback when using genetically modified iPSCs. Therefore, we have transduced iPSCs with lentiviral constructs overexpressing CDD from different constitutive promoter/enhancer elements and have investigated the effects of the ubiquitous chromatin opening element (UCOE) on transgene stability and CDD-mediated drug resistance in hematopoietically differentiated and naïve iPSCs.Materials/Methods: Murine iPSCs were transduced with 3rd-generation self-inactivating (SIN) lentiviral constructs overexpressing CDD and an IRES-coupled dTomato reporter from a truncated elongation factor 1α (EFS) or spleen focus-forming (SFFV) promoter/enhancer. Optionally, the UCOE site was cloned upstream of the respective promoter/enhancer. Transgene expression, Ara-C resistance and selection potential were investigated for naïve iPSCs and after differentiation along the hematopoietic lineage (d 0–8). Ara-C resistance was analyzed for colony-forming cells (d8-16). Expression of transgenic dTomato and CDD was measured by FACS, western blot and qRT-PCR. Bisulfite sequencing was performed to assess promoter methylation for the different lentiviral constructs. Results:Our studies demonstrated efficient transduction and stable EFS-driven CDD expression in undifferentiated iPSCs irrespective of the UCOE site. In contrast, SFFV-driven CDD expression was rapidly silenced. Although transgene expression levels were higher in UCOE.EFS.CDD- versus EFS.CDD-iPSCs (MFI: 89.5 vs 39.0), both, EFS.CDD- and UCOU.EFS.CDD-iPSCs were significantly protected against exposure to Ara-C (2000 nM/ 48 h) and were efficiently selected by continuous exposure to 2000 nM Ara-C (increase of dTomato+ cells from 5–8 % to 75–98 % within 16 days). No influence of CDD expression on iPSC morphology, growth characteristics, and expression of the pluripotency markers Oct4, Sox2, or Nanog was noted. Upon hematopoietic differentiation profound transgene silencing was observed in EFS.CDD-iPSCs. Silencing occurred during the first days of differentiation. Only 5–9% dTomato+ cells were observed on days 4 or 8, and reduced CDD expression levels were detected on days 4, 8, and 16 by Western blot and qRT-PCR analysis. Nevertheless, hematopoietic colony-forming units displayed significant resistance to Ara-C when compared to non-transduced controls. In contrast, UCOE.EFS.CDD-iPSCs only showed minor degrees of differentiation-induced transgene silencing with approx. 50% of the cells still expressing the dTomato transgene on day 8. Moreover, when subjected to clonogenic assays in the presence of 1000nM Ara-C, UCOE.EFS.CDD- in comparison to EFS.CDD-transduced cells exhibited significantly increased drug resistance (colony survival: 74±15 vs. 48±7%, p<0.05, n=3). In addition, bisulfite sequencing demonstrated significantly reduced CpG methylation in UCOE.EFS.CDD transduced cells upon hematopoietic differentiation. Conclusions:Our data suggest that Ara-C resistance in the hematopoietic system can be achieved by hematopoietic differentiation of CDD-overexpressing iPSCs. While EFS and SFFV promoter/enhancer elements upon hematopoietic differentiation are prone to epigenetic silencing, this may be overcome by the use of a UCOE element, which stabilizes transgene expression during hematopoietic differentiation and significantly reduces CpG methylation in regulatory elements of the provirus. Thus, our UCOE.EFS.CDD vector allows for long-term transgene expression in hematopoietic progeny of iPSCs, and hematopoietic differentiation of gene modified, patient-derived iPSCs may be suitable to increase the safety of drug resistance gene therapy in malignant diseases with manifestation in the hematopoietic system. Disclosures:No relevant conflicts of interest to declare.

Capsid-specific T-cell Responses to Natural Infections With Adeno-associated Viruses in Humans Differ From Those of Nonhuman Primates

Hepatic adeno-associated virus serotype 2 (AAV2)-mediated gene transfer failed to achieve sustained transgene product expression in human subjects. We formulated the hypothesis that rejection of AAV-transduced hepatocytes is caused by AAV capsid-specific CD8(+) T cells that become reactivated upon gene transfer. Although this hypothesis was compatible with clinical data, which showed a rise in circulating AAV capsid-specific T cells following injection of AAV vectors, it did not explain that AAV vectors achieved long-term transgene expression in rhesus macaques, which are naturally infected with AAV serotypes closely related to those of humans. To address this apparent contradiction, we tested human and rhesus macaque samples for AAV capsid-specific T cells by intracellular cytokine staining combined with staining for T-cell subset and differentiation markers. This highly sensitive method, which could provide a tool to monitor adverse T-cell responses in gene transfer trials, showed that AAV capsid-specific CD8(+) and CD4(+) T cells can be detected in blood of naturally infected humans and rhesus macaques. They are present at higher frequencies in rhesus macaques. Furthermore, T cells from humans and rhesus macaques exhibit striking differences in their differentiation status and in their functions, which may explain the disparate duration of AAV-mediated gene transfer in these two species.

The adeno-associated virus-mediated HSV-TK/GCV suicide system: a potential strategy for the treatment of bladder carcinoma

Novel treatment strategies such as gene therapy are warranted in view of the failure of current treatment approaches to cure a high percentage of patients with advanced bladder cancers. The emergence of cancer gene therapy potentially offers a number of exciting treatments. The majority of approaches involve strategies to suppress the function of activated oncogenes to restore the expression of functional tumour suppressor genes or to initiate tumour self-destruction. One gene therapy approach against tumours that holds great promise is suicide gene therapy. Herpes simplex virus thymidine kinase (HSV-TK) phosphorylates ganciclovir (GCV), which in turn interacts with cellular DNA polymerase and interferes with DNA synthesis to cause death of rapidly dividing cells. The development of an effective delivery system is absolutely critical to the usefulness and safety of gene therapy. At present, the adeno-associated virus (AAV) vector has the most promising potential in view of its non-pathogenicity, wide tropisms and long-term transgene expression in vivo. Gene therapy studies using different serotypes of recombinant AAV (rAAV) as delivery vehicles have proved rAAVs to be an effective modality of cancer gene therapy. In the present study, we investigated the suppression effect of AAV-mediated HSV-TK/GCV system on the bladder cancer cells and in mice xenograft models of bladder cancer. Our data demonstrate that rAAV-HSV-TK system controlled tumour cell growth and achieves strong antitumour efficacy in vivo. These findings provide a foundation for the development of potential targeted clinical therapies for bladder cancer in humans.

Induction of CD4 + CD25 + Foxp3 + T regulatory cells by dendritic cells derived from ILT3 lentivirus-transduced human CD34 + Cells

Immunoglobulin-like transcript 3 (ILT3) belongs to a family of inhibitory receptors with cytoplasmic immunoreceptor tyrosine based inhibitory motifs (ITIMs). Numerous studies have reported that increased ILT3 expression is associated with the tolerogenic properties of antigen-presenting cells (APCs) including dendritic cells (DCs). In this study, human CD34 + hematopoietic stem/progenitor cells (HPSCs) were transduced with self-inactivating lentiviral vector carrying the ILT3 gene, and then induced to differentiate into DCs. Long-term and sustained transgene expression were observed. Importantly, DCs differentiated from ILT3-transduced HPSCs expressed high levels of human ILT3 and acquired strong tolerogenic capacity. This effect was associated with markedly decreased expression of co-stimulatory molecules (CD80, CD86) and down-regulation of NF-κB. Functionally, ILT3 high DCs showed a reduced capacity to stimulate allogeneic T cell proliferation and increased the production of CD4 +CD25 +Foxp3 + T regulatory cells with immunosuppressive activity. These results demonstrate that DCs derived from ILT3-transduced human CD34 +HPSCs display tolerogenic properties to induce T regulatory cells in vitro.

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.

Therapeutic Applications of the PhiC31 Integrase System

The potential use of the ΦC31 integrase system in gene therapy opens up the possibilities of new treatments for old diseases. ΦC31 integrase mediates the integration of plasmid DNA into the chromsomes of mammalian cells in a sequence-specific manner, resulting in robust, long-term transgene expression. In this article, we review how ΦC31 integrase mediates transgene integration into the genomes of target cells and summarize the recent preclinical applications of the system to gene therapy. These applications encompass in vivo studies in liver and lung, as well as increasing ex vivo uses of the system, including in neural and muscle stem cells, in cord-lining epithelial cells, and for the production of induced pluripotent stem cells. The safety of the ΦC31 integrase system for gene therapy is evaluated, and its ability to provide treatments for hemophilia is discussed. We conclude that gene therapy strategies utilizing ΦC31 integrase offer great promise for the development of treatments in the future.

Helper-dependent canine adenovirus vector-mediated transgene expression in a neurodegenerative lysosomal storage disorder

Mucopolysaccharidosis type IIIA (MPS-IIIA) is a severe neurodegenerative lysosomal storage disorder caused by a deficiency of N-sulfoglucosamine sulfohydrolase (SGSH) activity with subsequent accumulation of partially-degraded heparan sulfate and other glycolipids. In this study, we have evaluated a gene therapy approach using a helper-dependent canine adenovirus vector that expresses human SGSH as a means of delivering sustained transgene expression to the brain. Initial testing in a mixed neural cell culture model demonstrated that the vector could significantly increase SGSH activity in transduced cells, resulting in near-normalization of heparan sulfate-derived fragments. While administration of vector by direct injection into the brain of adult MPS-IIIA mice enabled transgene expression for at least 8.5 months post-treatment, it was only in discrete areas of brain. Heparan sulfate storage was reduced in some regions following treatment, however there was no improvement in secondary neuropathological changes. These data demonstrate that helper-dependent canine adenovirus vectors are capable of neural transduction and mediate long-term transgene expression, but increased SGSH expression throughout the brain is likely to be required in order to effectively treat all aspects of the MPS-IIIA phenotype.

Non‐viral retinal gene therapy: a review

In the developed world, diseases of the retina are common causes of untreatable blindness. In many cases, a genetic component to the aetiology has been identified, making the development of gene-based treatments a logical long-term goal. The clinical strategy for retinal gene therapy broadly encompasses two distinct advantages over systemic drug delivery. First is that gene delivery can limit expression of a therapeutic protein to a specific target cell, which is rarely possible even with local drug delivery methods. Second, by delivering DNA that remains stable and non-degraded, gene expression and hence protein production could in theory be indefinite, obviating the need for repeated tablets or injections. Viruses have evolved distinct mechanisms, such as receptor mediated uptake and genomic integration, which efficiently encompass these two properties. For non-viral gene therapy approaches, however, nuclear localization and stable long-term transgene expression remain significant hurdles that need to be overcome. The challenge of non-viral gene therapy is therefore to harness current laboratory and molecular-based techniques to develop a man-made system that can approach the efficiency of a natural biological process. In the unique environment of the retina, this goal may not be insurmountable and would overcome the major limiting factor of adeno-associated viral vectors, which is the size of gene that can be delivered.

Adenovirus: Teaching an Old Dog New Tricks

Adenovirus was the first DNA virus to enter rigorous therapeutic development, largely because of its welldefined biology, its genetic stability, and its ease of large-scale production. Over the years, the virus has seen many aspects of development, ranging from its deployment as a vector for transgene delivery and supplementation and vaccination through to its use as an oncolytic agent. These developments have encompassed the use of wild-type adenoviruses for cancer therapy and as vaccines, oncolytic viruses with subtle deletions that are intended for functional complementation by the tumor phenotype or that are reliant on tumor-associated transcription factors, E1and E1,E3-deleted viruses for transgene delivery, through to high-capacity helper-dependent adenoviruses that are intended to deliver very large genetic cargoes with minimal provocation of antiviral immunity. Because adenovirus occurs (in humans) as 57 known serotypes grouped as 7 species (A–G), these diverse technological approaches can give rise to a vast range of therapeutic candidate viruses. It is, therefore, not surprising that adenovirus continues to occupy center stage in gene therapy engineering, and this issue of Human Gene Therapy presents a range of papers that illustrate the innovative approaches being pursued to maximize the utility of this versatile platform. It is widely accepted that poor delivery represents a limiting factor for most forms of in vivo gene therapy, particularly extrahepatic gene therapy, the only exceptions being where therapeutic viruses can be injected directly into the target site or where they can be introduced into target cells ex vivo. Delivery is very much a pharmaceutical concept, and optimizing delivery relies on measuring and improving the distribution and bioavailability of viral particles, as distinct from just measuring transduction, as that does not take account of unsuccessful delivery events. Accordingly, optimizing delivery requires elucidation of ‘‘particle kinetics,’’ and it is refreshing when a study reports efficient recovery of the number of viral particles that were initially injected. This is usually performed by quantitative PCR to measure viral genomes, and is generally possible only in very short time frames, as viral particles are rapidly catabolized in vivo. In this volume, Zhang and colleagues have prepared Ad5 with modified hexon protein, to prevent binding to factor X, leading to less uptake into hepatocytes in vivo and decreased toxicity. This innovative approach to decreasing hepatocyte toxicity is superior to the use of intracellular controls such as engineering the virus to include binding sites for hepatocytespecific microRNA, because it decreases the hepatocyte capture of viral particles and should have a positive effect on particle kinetics. This decreased hepatic toxicity is a definite advantage in mice, although we are still awaiting clinical data showing that the equivalent hepatic toxicity is a problem in humans. In addition, preventing capture of viral particles by Kupffer cells is likely to synergize with ablation of hepatocyte infection in extending plasma circulation of viral particles. Overcoming poor infection rates is an unusual challenge because adenovirus is rather promiscuous in its tropism. Nevertheless, to improve infection of refractory cells, Wang and colleagues have used a cationic liposome preparation to deliver genomes and thereby overcome the deficiencies of the coxsackievirus–adenovirus receptor (CAR) to which they attribute poor infection of the virus. Acute toxicity and rapid elimination of adenoviral particles after intravenous delivery are due to its interaction with the innate immune system, and the paper by Suzuki and colleagues shows that the cytosolic sensor protein NOD2 (nucleotide-binding and oligomerization domain-2) plays an important role in regulating the immediate response to adenoviral particles. It follows that NOD2 inhibitors might decrease acute inflammatory responses to adenovirus, and simultaneously perhaps increase circulation time. The adaptive immune response to viral particles and transgene products is also a major challenge, particularly for the use of adenovirus for long-term expression of transgenes. The observation by Seregin and colleagues that incorporating the complement inhibitor DAF (decay-accelerating factor) into the viral capsid can decrease immune provocation by both viral capsid proteins and transgenes may provide important insights into how viral biocompatibility can be enhanced. It may be possible to use this, or related, approaches both to decrease toxicity and to improve persistence of the vector and its cargo. Oncolytic virotherapy represents perhaps the most exciting avenue of adenovirus development; however, the absence of representative animal models has continually frustrated the study of oncolytic activity in the presence of a functioning immune system. The demonstration by Weaver and colleagues

Liver-Directed Gene Therapy with Helper-Dependent Adenoviral Vectors: Current State of the Art and Future Challenges

Successful liver-directed gene therapy has the potential to revolutionize medicine. Helper-dependent adenoviral vectors (HDAds) are devoid of all viral coding sequences and have shown tremendous potential for liver-direct gene therapy. In small and large animals, hepatic transduction with HDAd has resulted in high level, long-term transgene expression without chronic toxicity in a variety of disease models. Recent advancements in the large-scale manufacture of HDAd have permitted contemplation of clinical application. However, dose-dependent activation of the host innate inflammatory response remains an obstacle for clinical translation. Recent advancements in vector capsid modifications, immune modulation regimes, as well as novel routes of vector administration may yet permit clinical liver-directed gene therapy with HDAd.

Adeno-associated Viral Vectors for Correction of Inborn Errors of Metabolism: Progressing Towards Clinical Application

Gene therapy holds great promise for the treatment of inherited metabolic disease. Among different vector systems used to date, vectors based on adeno-associated virus (AAV) have shown great potential for systemic expression of therapeutic transgenes. The main advantages of AAV are the beneficial safety profile and the possibility to generate long-term transgene expression without the necessity for chromosomal integration. Successful transduction of hepatocytes in the absence of immunological complications has been achieved in a number of animal species, including mice, dogs and nonhuman primates. Despite this plethora of successful studies, clinical applications have been lagging. Up to date, one clinical trial for liver-directed gene transfer has been performed and results underscored the important role of neutralizing antibodies towards the AAV capsid and the generation of cytotoxic T cell responses against transduced hepatocytes. Recently, a wide variation of novel AAV serotypes has emerged that shows great promise for improved gene transfer efficiency. However, one important factor hampering clinical progress has been the large degree of variability in terms of transduction efficiency and transgene expression levels of different AAV serotypes and the subsequent difficulties in the selection of a single serotype for clinical development. The aim of this review is to critically reevaluate pre-clinical data obtained in animal models of metabolic diseases in light of the progress that has been achieved in liver-directed gene transfer using AAV vectors. Using this evidence-based rationale, we have selected AAV8 as the serotype that combines the most favorable features for clinical applications of hepatic gene transfer.

Engineered lentiviral vectors pseudotyped with a CD4 receptor and a fusogenic protein can target cells expressing HIV-1 envelope proteins

Lentiviral vectors (LVs) derived from human immunodeficiency virus type 1 (HIV-1) are promising vehicles for gene delivery because they not only efficiently transduce both dividing and non-dividing cells, but also maintain long-term transgene expression. Development of an LV system capable of transducing cells in a cell type-specific manner can be beneficial for certain applications that rely on targeted gene delivery. Previously it was shown that an inverse fusion strategy that incorporated an HIV-1 receptor (CD4) and its co-receptor (CXCR4 or CCR5) onto vector surfaces could confer to LVs the ability to selectively deliver genes to HIV-1 envelope-expressing cells. To build upon this work, we aim to improve its relatively low transduction efficiency and circumvent its inability to target multiple tropisms of HIV-1 by a single vector. We investigated a method to create LVs co-enveloped with the HIV-1 cellular receptor CD4 and a fusogenic protein derived from the Sindbis virus glycoprotein and tested its efficiency to selectively deliver genes into cells expressing HIV-1 envelope proteins. The engineered LV system yields a higher level of transduction efficiency and a broader tropism towards cells displaying the HIV-1 envelope protein (Env) than the previously developed system. Furthermore, we demonstrated in vitro that this engineered LV can preferentially deliver suicide gene therapy to HIV-1 envelope-expressing cells. We conclude that it is potentially feasible to target LVs towards HIV-1-infected cells by functional co-incorporation of the CD4 and fusogenic proteins, and provide preliminary evidence for further investigation on a potential alternative treatment for eradicating HIV-1-infected cells that produce drug-resistant viruses after highly active antiretroviral therapy (HAART).

Local Long-Term Expression of Lentivirally Delivered IL-10 in the Lung Attenuates Obliteration of Intrapulmonary Allograft Airways

Obliterative bronchiolitis (OB) is a form of chronic rejection after lung transplantation. Lentiviral vectors (LVs) facilitate long-term gene transduction in many tissues and organs. We hypothesized that lentiviral gene transfer of interleukin (IL)-10, a potent immune-modulating cytokine, to the lung could modulate the alloimmune responses in the lung after transplantation. C57BL6 mice received LVs encoding luciferase, enhanced green fluorescent protein (eGFP), or human IL-10 (huIL-10) through airways and underwent repeated bioluminescent imaging, immunofluorescence imaging, or ELISA of lung tissues, respectively. Luciferase activities peaked at day 7 and were stable after day 28 to over 15 months. eGFP staining demonstrated LV-mediated gene transduction mainly in alveolar macrophages. LV-huIL-10 delivery resulted in stable long-term expression of huIL-10 in the lung tissue (average 3.66 pg/mg at 1 year). Intrapulmonary allograft tracheal transplantation (BALBc→C57BL6) was used as a model of OB. LV-huIL-10 or LV-eGFP were delivered 7 days before transplantation and compared with no LV-transfection group. Allograft airways at day 28 were almost completely obliterated in all the groups. However, at day 42, allograft airways treated with LV-huIL-10 showed a spectrum of attenuation in airway fibrosis ranging from complete obliteration through bubble-like partial opening to complete patency with epithelial coverage in association with a significantly reduced obliteration ratio compared with the other groups (p<0.05). In conclusion, lentivirus-mediated gene transduction is useful in achieving long-term transgene expression in the lung. Long-term IL-10 expression has the potential to attenuate allograft airway obliteration. LV-mediated gene therapy could be a useful strategy to prevent or treat OB after lung transplantation.

Rescue, Amplification, and Large-Scale Production of Helper-Dependent Adenoviral Vectors

INTRODUCTIONThis protocol describes in detail the rescue, amplification, and large-scale production of helper-dependent adenoviral vectors (HDAds) for gene transfer and gene therapy.Production of HDAd can be divided into three parts: (1) rescue, which involves converting pHDAd (plasmid form) to HDAd (viral form); (2) amplification, in which the amount of HDAd is increased by serial coinfections (passages) of the producer cells with the HDAd and the helper virus; and (3) large-scale production to generate large quantities of HDAd. HDAds (also referred to as gutless, gutted, mini, fully deleted, high-capacity, Δ, pseudo) are deleted of all virus-coding sequences. HDAds retain the advantages of early-generation Ad vectors including high-efficiency in vivo transduction and high-level transgene expression. However, the absence of viral gene expression in transduced cells permits long-term transgene expression in the absence of chronic toxicity. Moreover, the deletion of the viral sequences permits a cloning capacity of ∼37 kb. This allows for the delivery of whole-genomic loci, multiple transgenes, and large cis-acting elements to enhance, prolong, and regulate transgene expression. In addition, because the vector genome exists episomally in transduced cells, the risks of germ-line transmission and insertional mutagenesis leading to oncogenic transformation are negligible.

Sustained, high transgene expression in liver with plasmid vectors using optimized promoter‐enhancer combinations

Plasmid-based gene therapy approaches often lack long-term transgene expression in vivo as a result of silencing or loss of the vector. One way to overcome these limitations is to combine nonsilenced promoters with strong enhancers. In the present study, we combine murine or human cytomegalovirus (CMV)-derived enhancer elements with the human elongation factor 1α (EF1α) promoter in a plasmid backbone devoid of potentially immunostimulating cytosine-guanine repeat sequences. Luciferase transgene activity was monitored in mouse liver after hydrodynamic plasmid delivery. Luciferase activity of a CMV-promoter driven plasmid rapidly declined within days, whereas the activity of the EF1α driven plasmid remained high for 2 weeks (murine enhancer) and detectable for > 80 days (human enhancer). Expression levels clearly correlated with higher plasmid copy number found in the liver at 2 months after gene delivery. Furthermore, we developed a novel synthetic CMV-EF1α hybrid promoter (SCEP) combining the high activity of CMV and sustained activity of EF1α promoter. The SCEP led to a constitutive three-fold increase in expression levels compared to the EF1α promoter in vivo. This novel combination of enhancer and promoter element with optimized plasmid backbones will pave the way for more efficient nonviral approaches in gene therapy.