Long-term Transgene Expression Research Articles

The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9–dependent innate immune responses in the liver

AbstractAlthough adeno-associated viral (AAV) vectors have been successfully used in hepatic gene transfer for treatment of hemophilia and other diseases in animals, adaptive immune responses blocked long-term transgene expression in patients on administration of single-stranded AAV serotype-2 vector. More efficient vectors have been developed using alternate capsids and self-complimentary (sc) genomes. This study investigated their effects on the innate immune profile on hepatic gene transfer to mice. A mild and transient up-regulation of myeloid differentiation primary response gene (88), TLR9, TNF-α, monocyte chemotactic protein-1, IFN-γ inducible protein-10, and IFN-α/β expression in the liver was found after single-stranded AAV vector administration, regardless of the capsid sequence. In contrast, scAAV vectors induced higher increases of these transcripts, upregulated additional proinflammatory genes, and increased circulating IL-6. Neutrophil, macrophage, and natural killer cell liver infiltrates were substantially higher on injection of scAAV. Some but not all of these responses were Kupffer cell dependent. Independent of the capsid or expression cassette, scAAV vectors induced dose-dependent innate responses by signaling through TLR9. Increased innate responses to scAAV correlated with stronger adaptive immune responses against capsid (but not against the transgene product). However, these could be blunted by transient inhibition of TLR9.

Adeno-associated viral vector-mediated transgene expression is independent of DNA methylation in primate liver and skeletal muscle.

Recombinant adeno-associated viral (rAAV) vectors can support long-term transgene expression in quiescent tissues. Intramuscular (IM) administration of a single-stranded AAV vector (ssAAV) in the nonhuman primate (NHP) results in a peak protein level at 2–3 months, followed by a decrease over several months before reaching a steady-state. To investigate transgene expression and vector genome persistence, we previously demonstrated that rAAV vector genomes associate with histones and form a chromatin structure in NHP skeletal muscle more than one year after injection. In the mammalian nucleus, chromatin remodeling via epigenetic modifications plays key role in transcriptional regulation. Among those, CpG hyper-methylation of promoters is a known hallmark of gene silencing. To assess the involvement of DNA methylation on the transgene expression, we injected NHP via the IM or the intravenous (IV) route with a recombinant ssAAV2/1 vector. The expression cassette contains the transgene under the transcriptional control of the constitutive Rous Sarcoma Virus promoter (RSVp). Total DNA isolated from NHP muscle and liver biopsies from 1 to 37 months post-injection was treated with sodium bisulfite and subsequently analyzed by pyrosequencing. No significant CpG methylation of the RSVp was found in rAAV virions or in vector DNA isolated from NHP transduced tissues. Direct de novo DNA methylation appears not to be involved in repressing transgene expression in NHP after gene transfer mediated by ssAAV vectors. The study presented here examines host/vector interactions and the impact on transgene expression in a clinically relevant model.

Clinical potential and challenges of using genetically modified cells for articular cartilage repair

Articular cartilage defects do not regenerate. Transplantation of autologous articular chondrocytes, which is clinically being performed since several decades, laid the foundation for the transplantation of genetically modified cells, which may serve the dual role of providing a cell population capable of chondrogenesis and an additional stimulus for targeted articular cartilage repair. Experimental data generated so far have shown that genetically modified articular chondrocytes and mesenchymal stem cells (MSC) allow for sustained transgene expression when transplanted into articular cartilage defects in vivo. Overexpression of therapeutic factors enhances the structural features of the cartilaginous repair tissue. Combined overexpression of genes with complementary mechanisms of action is also feasible, holding promises for further enhancement of articular cartilage repair. Significant benefits have been also observed in preclinical animal models that are, in principle, more appropriate to the clinical situation. Finally, there is convincing proof of concept based on a phase I clinical gene therapy study in which transduced fibroblasts were injected into the metacarpophalangeal joints of patients without adverse events. To realize the full clinical potential of this approach, issues that need to be addressed include its safety, the choice of the ideal gene vector system allowing for a long-term transgene expression, the identification of the optimal therapeutic gene(s), the transplantation without or with supportive biomaterials, and the establishment of the optimal dose of modified cells. As safe techniques for generating genetically engineered articular chondrocytes and MSCs are available, they may eventually represent new avenues for improved cell-based therapies for articular cartilage repair. This, in turn, may provide an important step toward the unanswered question of articular cartilage regeneration.

Generation of Replication-Competent and -Defective HSV Vectors

Engineering effective vectors has been crucial to the efficient delivery and expression of therapeutic gene products in vivo. Among these, HSV-1 represents an excellent candidate vector for delivery to the peripheral and central nervous systems. The natural biology of HSV-1 includes the establishment of a lifelong latent state in neurons in which the viral genome persists as an episomal molecule. Genomic HSV vectors can be produced that are completely replication-defective, nontoxic, and capable of long-term transgene expression. Herpes simplex virus (HSV) vectors are constructed by using a replication-deficient vector backbone (TOZ.1) for homologous recombination with a shuttle plasmid containing a cassette expressing the gene of interest inserted into the UL41 gene sequence. The TOZ.1 vector expresses a reporter gene (lacZ) in the UL41 locus, such that recombination of the transgenic cassette into the UL41 locus results in the loss of the reporter gene activity. The TOZ.1 vector also contains a unique PacI endonuclease site for digestion of parental viral DNA that substantially reduces the nonrecombinant background. Following homologous recombination of the shuttle plasmid into the PacI-digested TOZ.1 genome, the recombinants are identified as clear plaques. After three rounds of limiting dilution analysis, the structure of the recombinants can be confirmed by Southern blot or by polymerase chain reaction (PCR) analysis.

Single intra-articular injection of adeno-associated virus results in stable and controllable in vivo transgene expression in normal rat knees

To test the hypothesis that invivo transgene expression mediated by single intra-articular injection of adeno-associated virus serotype 2 (AAV2) persists within intra-articular tissues 1 year post-injection and can be externally controlled using an AAV2-based tetracycline-inducible gene regulation system containing the tetracycline response element (TRE) promoter. Sprague Dawley rats received intra-articular injections of AAV2-cytomegalovirus (CMV)-enhanced green fluorescent protein (GFP) and AAV2-CMV-luciferase (Luc) into their right and left knees, respectively. Luciferase expression was evaluated over 1 year using bioluminescence imaging. After sacrifice, tissues were analyzed for GFP+ cells by fluorescent microscopy. To study external control of intra-articular AAV-transgene expression, another set of rats was co-injected with AAV2-TRE-Luc and AAV2-CMV-reverse-tetracycline-controlled transactivator (rtTA) into the right knees, and AAV2-CMV-Luc and AAV2-CMV-rtTA into the left knees. Rats received oral doxycycline (Dox), an analog of tetracycline, for 7 days. Luciferase expression was assessed by bioluminescence imaging. Luciferase expression was localized to the injected joint and persisted throughout the 1-year study period. Abundant GFP+ cells were observed within intra-articular soft tissues. Transgene expression in AAV2-TRE-Luc injected joints was upregulated by oral administration of Dox, and downregulated following its removal, at 14 days and 13 months post-AAV injection. This longitudinal invivo study shows that sustained and stable AAV-mediated intra-articular transgene expression can be achieved through a single intra-articular injection and can be controlled using a tetracycline-controlled inducible AAV system in a normal rat knee model. Highly regulatable long-term intra-articular transgene expression is of potential clinical utility for development of treatment strategies for chronic intra-articular disease processes such as inflammatory and degenerative arthritis.

Infectious delivery and long-term persistence of transgene expression in the brain by a 135-kb iBAC-FXN genomic DNA expression vector

Novel gene-based therapies for disease will depend in many cases on long-term persistent transgene expression. To develop gene therapy strategies for Friedreich's ataxia (FRDA), we have examined the persistence of transgene expression in the brain in vivo provided by the entire 135 kb FXN genomic DNA locus delivered as an infectious bacterial artificial chromosome (iBAC) herpes simplex virus type 1 (HSV-1)-based vector injected in the adult mouse cerebellum. We constructed genomic DNA-reporter fusion vectors carrying a complete 135 kb FXN genomic locus with an insertion of the Escherichia coli lacZ gene at the ATG start codon (iBAC-FXN-lacZ). SHSY5Y human neuroblastoma cells transduced by iBAC-FXN-lacZ showed high efficiency of vector delivery and LacZ expression. Direct intracranial injection of iBAC-FXN-lacZ into the adult mouse cerebellum resulted in a large number of easily detectable transduced cells, with LacZ expression driven by the FXN genomic locus, which persisted for at least 75 days. Green fluorescent protein expression driven from the same vector but by the strong HSV-1 IE4/5 promoter was transient. Our data demonstrate for the first time sustained transgene expression in vivo by infectious delivery of a genomic DNA locus >100 kb in size. Such an approach may be suitable for gene rescue strategies in neurological disease, such as FRDA.

Helper-dependent adenoviral vectors for liver-directed gene therapy

Helper-dependent adenoviral (HDAd) vectors devoid of all viral-coding sequences are promising non-integrating vectors for liver-directed gene therapy because they have a large cloning capacity, can efficiently transduce a wide variety of cell types from various species independent of the cell cycle and can result in long-term transgene expression without chronic toxicity. The main obstacle preventing clinical applications of HDAd for liver-directed gene therapy is the host innate inflammatory response against the vector capsid proteins that occurs shortly after intravascular vector administration resulting in acute toxicity, the severity of which is dependent on vector dose. Intense efforts have been focused on elucidating the factors involved in this acute response and various strategies have been investigated to improve the therapeutic index of HDAd vectors. These strategies have yielded encouraging results with the potential for clinical translation.

Optimization of Lentiviral Vectors Generation for Biomedical and Clinical Research Purposes: Contemporary Trends in Technology Development and Applications

Classical non-viral methods of gene transfer, such as chemical transfection, have met with limited success of instillation of genetic material into non-proliferating cells in vitro. Among the different kinds of viral vectors, Lentiviral vectors (LVs) have emerged as robust and versatile tool for ex vivo and in vivo gene delivery into multiple cell types including non-dividing cells such as neurons. The capacity of LVs to maintain stable, long-term transgene expression and the substantial flexibility in the design of the expression cassettes account for their increasing use in various pre-clinical and clinical applications. Additionally, LVs have been hugely successful in reprogramming induced pluripotent stem cells (iPSCs). Recent development using LVs in conjunction with a Cre-Lox based reversible system has opened up many new possibilities towards therapeutic application of iPSC technology in various clinical settings. Moreover, improvements in term of biosafety and efficacy, achieved either by modifying the vector design or by involving integration-deficient LVs (IDLVs), have important implications for adoption of LV as the vector of choice for clinical trials. Several human gene therapy clinical trials evaluating the use of LVs for treatment: of human diseases such as Parkinson's disease, β-thalassemia, X-linked adrenoleukodystrophy (ALD), and AIDS are currently ongoing. This review will describe the state of the art achieved by LV technology, its impact on biomedical research, and implications to human clinical trials as therapeutic gene delivery vehicle for a wide range of infectious and genetic diseases.

Interactions Between AAV-2 and HSV-1: Implications for Hybrid Vector Design

Herpes simplex virus type 1 (HSV-1)-based amplicon vectors have a transgene capacity of up to 150 kbp and can efficiently transduce many different cell types in culture and in vivo without causing cytopathic effects. However, these vectors do not support long-term transgene expression. Adeno-associated virus type 2 (AAV-2) has the capacity to integrate its genome into a specific site on human chromosome 19, but AAV-2-derived gene therapy vectors have a transgene capacity of only 4.5 kb. To combine the large transgene capacity of HSV-1 with the potential for site-specific genomic integration and long-term transgene expression of AAV-2, HSV/AAV hybrid vectors have been developed. This review describes the design, applications and limitations of these hybrid vectors. However, as HSV-1 is a full helper virus for AAV-2 replication, the main focus is the analysis of the molecular mechanisms of interaction between the two viruses. The knowledge of these interactions will have direct implications on the design of novel HSV/AAV hybrid vectors.

Targeting Neurons of Rat Nucleus Tractus Solitarii with the Gene Transfer Vector Adeno-Associated Virus Type 2 to Up-Regulate Neuronal Nitric Oxide Synthase

Adeno-associated virus (AAV) has distinct advantages over other viral vectors in delivering genes of interest to the brain. AAV mainly transfects neurons, produces no toxicity or inflammatory responses, and yields long-term transgene expression. In this study, we first tested the hypothesis that AAV serotype 2 (AAV2) selectively transfects neurons but not glial cells in the nucleus tractus solitarii (NTS) by examining expression of the reporter gene, enhanced green fluorescent protein (eGFP), in the rat NTS after unilateral microinjection of AAV2eGFP into NTS. Expression of eGFP was observed in 1-2 cells in the NTS 1day after injection. The number of transduced cells and the intensity of eGFP fluorescence increased from day 1 to day 28 and decreased on day 60. The majority (92.9±7.0%) of eGFP expressing NTS cells contained immunoreactivity for the neuronal marker, protein gene product 9.5, but not that for the glial marker, glial fibrillary acidic protein. We observed eGFP expressing neurons and fibers in the nodose ganglia (NG) both ipsilateral and contralateral to the injection. In addition, eGFP expressing fibers were present in both ipsilateral and contralateral nucleus ambiguus (NA), caudal ventrolateral medulla (CVLM) and rostral ventrolateral medulla (RVLM). Having established that AAV2 was able to transduce a gene into NTS neurons, we constructed AAV2 vectors that contained cDNA for neuronal nitric oxide synthase (nNOS) and examined nNOS expression in the rat NTS after injection of this vector into the area. Results from RT-PCR, Western analysis, and immunofluorescent histochemistry indicated that nNOS expression was elevated in rat NTS that had been injected with AAV2nNOS vectors. Therefore, we conclude that AAV2 is an effective viral vector in chronically transducing NTS neurons and that AAV2nNOS can be used as a specific gene transfer tool to study the role of nNOS in CNS neurons.

Intrathecal Injection of Helper-Dependent Adenoviral Vectors Results in Long-Term Transgene Expression in Neuroependymal Cells and Neurons

Helper-dependent adenoviral (HDAd) vectors are devoid of all viral genes and result in long-term transgene expression in the absence of chronic toxicity. Because of their ability to infect post-mitotic cells, including cells of the central nervous system, HDAd vectors are particularly attractive for brain-directed gene therapy. In this study, we show that intrathecal injection of HDAd results in extensive transduction of ependymal cells and sustained expression of the transgene up to 1 year post-administration. We also demonstrate, for the first time, the ability of HDAd injected by this route of delivery to transduce neuronal cells. The transduced neuroepithelial cells can be potentially used to secrete therapeutic proteins into the cerebrospinal fluid and provide them via cross-correction to nontransduced cells. Targeting of neuronal cells and long-term transgene expression make this approach attractive for the treatment of several neurologic diseases.

RETRACTED: Activation of the NF-κB pathway by adeno-associated virus (AAV) vectors and its implications in immune response and gene therapy

Because our in silico analysis with a human transcription factor database demonstrated the presence of several binding sites for NF-κB, a central regulator of cellular immune and inflammatory responses, in the adeno-associated virus (AAV) genome, we investigated whether AAV uses NF-κB during its life cycle. We used small molecule modulators of NF-κB in HeLa cells transduced with recombinant AAV vectors. VP16, an NF-κB activator, augmented AAV vector-mediated transgene expression up to 25-fold. Of the two NF-κB inhibitors, Bay11, which blocks both the canonical and the alternative NF-κB pathways, totally ablated transgene expression, whereas pyrrolidone dithiocarbamate, which interferes with the classical NF-κB pathway, had no effect. Western blot analyses confirmed the abundance of the nuclear p52 protein component of the alternative NF-κB pathway in the presence of VP16, which was ablated by Bay11, suggesting that AAV transduction activates the alternative NF-κB pathway. In vivo, hepatic AAV gene transfer activated the canonical NF-κB pathway within 2 h, resulting in expression of proinflammatory cytokines and chemokines (likely reflecting the sensing of viral particles by antigen-presenting cells), whereas the alternative pathway was activated by 9 h. Bay11 effectively blocked activation of both pathways without interfering with long-term transgene expression while eliminating proinflammatory cytokine expression. These studies suggest that transient immunosuppression with NF-κB inhibitors before transduction with AAV vectors should lead to a dampened immune response, which has significant implications in the optimal use of AAV vectors in human gene therapy.

Recombinant Adeno-Associated Virus-Mediated In Utero Gene Transfer Gives Therapeutic Transgene Expression in the Sheep

Somatic in utero gene therapy aims to treat congenital diseases where pathology develops in perinatal life, thereby preventing permanent damage. The aim of this study was to determine whether delivery of self-complementary (sc) adeno-associated virus (AAV) vector in utero would provide therapeutic long-term transgene expression in a large animal model. We performed ultrasound-guided intraperitoneal injection of scAAV2/8-LP1-human Factor IX (hFIX)co (1 × 10(12) vector genomes/kg) in early (n = 4) or late (n = 2) gestation fetal sheep. The highest mean hFIX levels were detected 3 weeks after injection in late gestation (2,055 and 1,687.5 ng/ml, n = 2) and 3 days after injection in early gestation (435 ng/ml, n = 1). Plasma hFIX levels then dropped as fetal liver and lamb weights increased, although low levels were detected 6 months after late gestation injection (75 and 52.5 ng/ml, n = 2). The highest vector levels were detected in the fetal liver and other peritoneal organs; no vector was present in fetal gonads. hFIX mRNA was detectable only in hepatic tissues after early and late gestation injection. Liver function tests and bile acid levels were normal up to a year postnatal; there was no evidence of liver pathology. No functional antibodies to hFIX protein or AAV vector were detectable, although lambs mounted an antibody response after injection of hFIX protein and Freund's adjuvant. In conclusion, hFIX expression is detectable up to 6 months after delivery of scAAV vector to the fetal sheep using a clinically applicable method. This is the first study to show therapeutic long-term hFIX transgene expression after in utero gene transfer in a large animal model.

Strategies for selection marker-free swine transgenesis using the Sleeping Beauty transposon system

Swine transgenesis by pronuclear injection or cloning has traditionally relied on illegitimate recombination of DNA into the pig genome. This often results in animals containing concatemeric arrays of transgenes that complicate characterization and can impair long-term transgene stability and expression. This is inconsistent with regulatory guidance for transgenic livestock, which also discourages the use of selection markers, particularly antibiotic resistance genes. We demonstrate that the Sleeping Beauty (SB) transposon system effectively delivers monomeric, multi-copy transgenes to the pig embryo genome by pronuclear injection without markers, as well as to donor cells for founder generation by cloning. Here we show that our method of transposon-mediated transgenesis yielded 38 cloned founder pigs that altogether harbored 100 integrants for five distinct transposons encoding either human APOBEC3G or YFP-Cre. Two strategies were employed to facilitate elimination of antibiotic genes from transgenic pigs, one based on Cre-recombinase and the other by segregation of independently transposed transgenes upon breeding.

Helper-Dependent Adenoviral Vectors

Helper-dependent adenoviral vectors are devoid of all viral coding sequences, possess a large cloning capacity, and can efficiently transduce a wide variety of cell types from various species independent of the cell cycle to mediate long-term transgene expression without chronic toxicity. These non-integrating vectors hold tremendous potential for a variety of gene transfer and gene therapy applications. Here, we review the production technologies, applications, obstacles to clinical translation and their potential resolutions, and the future challenges and unanswered questions regarding this promising gene transfer technology.

Lifelong reporter gene imaging in the lungs of mice following polyethyleneimine-mediated sleeping-beauty transposon delivery

Polyethyleneimine (PEI) is a cationic polymer that is effective in gene delivery in vivo. Plasmid DNA incorporating the Sleeping-Beauty (SB) transposon has been shown to induce long-term transgene expression in mouse lungs after PEI-mediated delivery. In the current report, we followed the reporter gene expression mediated by PEI/SB delivery in lungs of mice using the non-invasive bioluminescent imaging (BLI) technology. After delivery, the reporter gene signal showed a rapid decay in the first two weeks to a nearly undetectable level, but then the signal augmented gradually in the following weeks and finally reached a stable level that maintained until the natural death of animals. The stabilization of transgene expression is associated with the multiplication of a small number of PEI/SB-labeled alveolar cells, which proliferated both under normal conditions and in response to acute local injury for epithelia repair, and may play a role in long-term homeostatic maintenance in alveoli. The data presented here suggests that systemic delivery of PEI/SB induces stable transfection specifically in a small population of alveolar progenitor cells. The technique provides a promising platform for future research in distal lung biology and tissue regenerative therapy.

Naturally-Occurring Antibodies to Human AAV In Sheep: A New Large Animal Model for Immune Aspects of AAV Gene Transfer.

AbstractAbstract 3762AAV vectors have received a great deal of attention for clinical gene therapy (GT), since they transduce many mitotic and quiescent cells and mediate long-term transgene expression. Unfortunately, many of the serotypes of AAV commonly employed in GT procedures ubiquitously infect humans, generating pre-existing immunity against the AAV capsid proteins that precludes efficient transduction or induces CTL responses to the transduced target tissue. At present, highly successful animal studies have not translated into clinical success in humans, due, at least in part, to the paucity of animals which harbor endogenous antibodies which recognize and bind AAV-2 and other AAV serotypes for which humans are the natural host. Sheep have long been used as a model to study a broad range of disease states, and a high degree of clinical predictability has consistently been observed. We therefore examined whether sheep possess antibodies to AAV and could thus serve as a much-needed preclinical model system for evaluating AAV-based GT. ELISAs were performed on sera from a panel of 6 healthy Merino-Rambouillet sheep using AAV-1,-2,-5,-6,-8, -9 particles as the antigen. Our results demonstrate that sheep naturally harbor antibodies to all 6 AAV serotypes tested, yet the titers against the different serotypes varied greatly from sheep-to-sheep. While one sheep exhibited very high level (>2300ng/ml) IgG against all 6 AAV serotypes tested, others exhibited moderate/low (>350ng/ml) IgG against all 6 AAV serotypes, and still others exhibited moderate/low level IgG against only 3–4 of the tested serotypes. Despite these differences, all sheep harbored detectable antibodies to AAV 2, 5, & 8. A luciferase-based neutralizing antibody (NAB) assay was then performed on sera from 3 of the sheep exhibiting the highest titer IgG against AAV 2, 8, & 9 to assess the clinical significance of these antibodies in the context of AAV-based GT. All 3 animals harbored relatively high titer (1:100-1:316) NAB to AAV 2, but only 1 animal harbored significant NAB titers against AAV 8 & 9 (1:31, and 1:100, respectively). B cell epitope mapping of these 3 animals with a library of peptides derived from the capsids of AAV 2, 5, 8, and 9 revealed that each individual sheep harbored antibodies recognizing from 17 to 50 of the various capsid-derived peptides, some of which were common to all capsids, and some of which were unique to specific AAV serotypes. Importantly, many of the identified capsid epitopes have also been shown to be recognized by antibodies present in human patients with existing AAV immunity. To our knowledge, this is the first report of an animal disease model harboring naturally occurring functional antibodies to serotypes of human AAV commonly employed as GT vectors. The close parallels between human and sheep physiology, coupled with our recent re-establishment of sheep with severe hemophilia A with a null mutation in the FVIII gene and the presence of these antibodies, suggest that sheep may represent an ideal large animal model system in which to study GT in the context of pre-existing immunity to AAV, and to develop novel strategies for circumventing this immunologic barrier. Disclosures:No relevant conflicts of interest to declare.

Suppression of bladder cancer growth in mice by adeno-associated virus vector-mediated endostatin expression

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. Testing of the hypothesis that blocking the angiogenic switch may keep tumour growth in check has been facilitated by the discovery of endogenous inhibitors of angiogenesis and has also added another research dimension to the field of cancer gene therapy. Consequently, the concept of targeting the tumour vasculature with anti-angiogenic agents has emerged as an attractive new strategy in the treatment of cancer. Targeted biological therapies that selectively interfere with tumour angiogenesis could improve survival among patients with bladder cancer. Endostatin is a tumour-derived angiogenesis inhibitor and is the first endogenous inhibitor of angiogenesis to be indentified in a matrix protein. Gene therapy represents an attractive approach to treat cancers and other chronic diseases. 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, an IgG fragment was inserted at the start of the sequence coding for endostatin with the aim of enabling continuous secretion of endostatin the serum. We also investigated the suppression effect of AAV-mediated endostatin expression on endothelial cells and in mice xenograft models of bladder cancer. Our data demonstrates that rAAV-endostatin controlled tumour cell growth and achieves strong anti-tumour efficacy in vivo.

RD114 envelope proteins provide an effective and versatile approach to pseudotype lentiviral vectors

Lentiviral vectors derived from the HIV-1 genome offer great promise for gene therapy due to their ability to transduce non-dividing cells and sustain long-term expression of transgenes. The majority of current lentiviral vectors are pseudotyped with the vesicular stomatitis viral envelope (VSV-G). VSV-G equips lentiviral vectors with a broad host cell tropism and increased stability. Increased particle stability enables viral supernatants to be concentrated by high-speed centrifugation to enhance their infectivity. Despite its efficacy, VSV-G is cytotoxic - a feature that prohibits the development of stable cell lines that constitutively express this envelope. Therefore, non-toxic envelope proteins are being investigated. RD114 is an attractive alternative because it also provides increased particle stability and its receptor is widely expressed on hematopoietic stem cells (HSCs). In this study, the packaging efficiency of three envelope proteins, RD114, RDpro and VSV-G, were evaluated with two lentiviral vectors (TRIP GFP and HPV-402). RDpro is an RD114-HIV chimera designed to pseudotype lentiviral vectors more efficiently. In transient systems, VSV-G generated titers of 10(8) and 10(7) viral particles/mL for TRIP GFP and HPV-402. RDpro possessed titers of 10(7) and 10(6), while RD114 titers were one log lower for each vector. Despite having relatively lower titers, RD114 proteins are less toxic; this was demonstrated in the extension of transient transfection reactions from 48 to 96 h. VSV-G transfections are generally limited to 48 h. In regard to gene therapy applications, we show that RDpro supernatants efficiently transduce peripheral blood HSCs. The versatility of RD114 envelopes was again demonstrated by using a 'mixed' expression system; composed of stably expressed RD114 envelope proteins to pseudotype lentiviral vectors generated in trans (titer range 10(3)-10(5)). Our data show that RD114 envelope proteins are effective and versatile constructs that could prove to be essential components of therapeutic lentiviral gene transfer systems.

Preclinical Studies on Specific Gene Therapy for Recessive Retinal Degenerative Diseases

Inherited retinal diseases are non-lethal and have a wide level of genetic heterogeneity. Many of the genes involved have now been identified and their function elucidated, providing a major step towards the development of gene-based treatments. The most widely used vectors for ocular gene delivery are based on adeno-associated virus (AAV) because they mediate long-term transgene expression in a variety of retinal cell types and elicit minimal immune responses. Extensive preclinical evaluation of gene transfer strategies in small and large animal models is key to the development of successful gene-based therapies for the retina. These preclinical studies have already allowed the field to reach the point where gene therapy to treat inherited blindness has been brought to clinical trial. In this manuscript, we focus on recombinant AAV-mediated specific gene therapy for recessive retinal degenerative diseases we describe the preclinical studies for the treatment of retinal degeneration caused by retinal pigmented epithelium (RPE) cells or photoreceptor defects and the immune response induced by retinal rAAV gene transfer.