Development of adenovirus hybrid vectors for Sleeping Beauty transposition in large mammals

The Sleeping Beauty (SB) transposase system for somatic integration offers great potential for in vivo gene therapeutic applications and genome engineering. Until recently, however, efficacy of SB transposase as a gene transfer vector especially in large animals was lacking. Herein, we report about the newest viral vector development for delivery of the SB transposase system into large mammals. Over the past decade various hyperactive versions of SB transposase and advanced adenovirus vectors enabling efficient and safe delivery of transgenes in vivo were developed. Already several years ago it was demonstrated that adenovirus vectors can be used for delivery of the SB transposase system into murine liver. Our newest study showed for the first time that a hyperactive transposase system delivered by high-capacity adenoviral vectors can result in somatic integration of exogenous DNA in canine liver, facilitating stabilized transgene expression and phenotypic correction for up to three years in a canine model of human disease. In this review we discuss safety issues and further improvements of this adenovirus based hybrid vector system for somatic integration. In the future this approach paves new paths towards the possible cure of human genetic diseases and novel strategies for in vivo genome engineering in large mammals.     

Stable transduction of large DNA by high-capacity adeno-associated virus/adenovirus hybrid vectors

Viral vectors with high cloning capacity and host chromosomal integration ability are in demand for the efficient and permanent genetic modification of target cells with large DNA molecules. We have generated a hybrid gene transfer vehicle consisting of recombinant adeno-associated virus (AAV) replicative intermediates packaged in adenovirus (Ad) capsids. This arrangement allows cell cycle-independent nuclear delivery of recombinant AAV genomes with lengths considerably above the maximum size (i.e., 4.7 kb) that can be accommodated within AAV capsids. Here we show that high-capacity AAV/Ad hybrid vector gene transfer mediates cellular genomic integration of large fragments of foreign DNA and accomplishes stable long-term transgene expression in rapidly proliferating cells. Southern blot and polymerase chain reaction analyses of chromosomal DNA extracted from clones of stably transduced cells revealed that most of them contained a single copy of the full-length hybrid vector genome with AAV inverted terminal repeat (ITR) sequences at both ends. The high-capacity AAV/Ad hybrid vector system can thus be used for the transfer and expression of transgenes that cannot be delivered by conventional integrating viral vectors.     

Development of a recombinant adenovirus vector production system free of replication-competent adenovirus by utilizing a packaging size limit of the viral genome

In a conventional adenovirus (Ad) vector production method using 293 cells, homologous recombination between Ad vector DNA and 293 cell-derived Ad E1 DNA occurs with low efficiency, resulting in the generation of replication-competent adenovirus (RCA). RCA can induce the spread of replication-incompetent Ad vectors, leading to unexpected tissue damage. In order to overcome this problem, we developed an Ad vector production system free of RCA generation by utilizing the Ad packaging size limit of the viral genome. It is well known that up to approximately 105% (37.7 kb) of the wild-type genome (35.9 kb) can be packaged in the Ad virion. We designed the Ad vector genome by insertion of a transgene expression cassette into the E3 region, such that homologous recombination between the Ad vector DNA and 293 cell-derived Ad E1 DNA would produce an Ad vector genome that exceeds in the size of the packaging limit. In accord with our strategy, no RCA generation was observed during the passages when we used the E1 (3.2kb)-deleted Ad vectors containing a more than 3.0-kb transgene expression cassette in the E3 region. In contrast, the E1 (3.2kb)-deleted Ad vectors, which retain 37.7 kb of the viral genome and have an insertion of a 2.1-kb transgene expression cassette in the E3 region, generated RCA, although RCA derived from this Ad vector exceeded the packaging size limit (105.0%). These results suggest that RCA generation can be avoided when the genome size of RCA is more than 108.3% (38.9 kb) of the wild-type Ad genome. This Ad vector production system generates safe, easy, and efficient Ad vector stock for both basic study as well as clinical research.     

慢病毒载体介导的转基因整合位点研究方法

慢病毒载体整合到宿主细胞的染色体上可以长期稳定表达,目前已成为基因治疗和转基因动物载体研究的热点.慢病毒载体整合的位置效应是影响外源基因表达的重要因素,慢病毒载体整合位点的研究是探索外源基因整合机制的手段之一.转基因整合位点研究的方法主要有5种:荧光原位杂交、个体基因组文库筛选法、反向PCR、接头PCR、锚定PCR.近来的研究后发现整合位点之间可能存在一些共同特征.     

The role of adipose-derived stem cells engineered with the persistently expressing hybrid baculovirus in the healing of massive bone defects

Massive segmental defects arising from trauma or tumor resection remain a challenging clinical problem. To repair large, segmental bone defects using adipose-derived stem cells (ASCs) which alone cannot heal massive defects, we hypothesized that sustained expression of factors promoting osteogenesis (BMP2) and angiogenesis (VEGF) provides continuous stimuli to augment the healing. Baculovirus is a vector for gene delivery into stem cells, but it only mediates transient expression. Therefore we developed a dual system whereby one baculovirus expressed FLP recombinase (BacFLP) while the other hybrid baculovirus harbored an Frt-flanking transgene cassette. Within the ASCs transduced with BacFLP and the hybrid baculovirus, the transduction efficiency reached 98% and the FLP/Frt-mediated recombination efficiency approached 46%, leading to cassette excision off the baculovirus genome, enabling transgene persistence in episomal form and prolonging the expression to >28 days. ASCs engineered by the conventional baculovirus transiently expressing BMP2/VEGF (S group) only healed the critical-size (10mm) segmental femoral bone defects in 40% of New Zealand White rabbits at 12 weeks post-implantation, whereas ASCs engineered by the hybrid vectors persistently expressing BMP2/VEGF (L group) healed the critical-size defects in 12 out of 12 animals in 8 weeks. Compared with the S group, the L group not only accelerated the healing, but also ameliorated the bone quality (metabolism, volume, density, mechanical properties) and angiogenesis, thereby attesting our hypothesis that persistent BMP2/VEGF expression is essential. Use of ASCs engineered by the hybrid BV vector thus holds promise to treat massive segmental defects necessitating sustained stimuli.     

Efficient delivery and stable gene expression in a hematopoietic cell line using a chimeric serotype 35 fiber pseudotyped helper-dependent adenoviral vector

Certain human cell populations have remained difficult to infect with human adenovirus (Ad) serotype 5 because of their lack of coxsackievirus B-adenovirus receptor (CAR). Native adenovirus fiber compositions, although diverse, cannot infect all tissue types. Recently, a chimeric Ad5/35 fiber was created, which displays an altered tropism from Ad5. We incorporated this chimeric fiber into a helper-dependent (HD) adenovirus vector system and compared HD to E1-deleted (E1Delta) vectors by transgene expression, cell transduction efficiency, and cytotoxicity. K562 cells were infected approximately 50 times more efficiently with the chimeric Ad5/35 fiber compared with the Ad5 fiber. Short-term transgene expression was sustained longer from HD Ad5/35 than E1Delta Ad5/35 vector after in vitro infection of actively dividing K562 cells. Rapid loss of transgene expression from E1Delta Ad5/35 infection was not due to the loss of vector genomes, as determined by quantitative real-time PCR (QRT-PCR), or cytotoxicity, but rather through a putative silencing mechanism.     

A new vector system with inducible E2a cell line for production of higher titer and safer adenoviral vectors

Adenoviral vectors have been used in gene therapy and for vaccination. The major concerns with using adenoviral vectors are the pathogenic potential of the virus backbone and the generation of replication-competent adenovirus that may replicate in an uncontrolled manner, especially in immunocompromised patients. It is important to develop new vectors that are safer for clinical trials while maintaining high titer and efficient transduction. A new adenovirus vector production system was developed, which includes several vector backbone plasmids deleted for E2a and a new cell line expressing both E1 and E2a. The new cell line with the tTA-inducible E2a expression cassette can significantly increase the titer of E1/E2a-deleted vectors by four to five orders of magnitude upon withdrawal of tetracycline. Furthermore, there is no sequence overlap between the vector and the cellular DNA within the E2a open reading frame and downstream, making the generation of virus with wild-type E2a through homologous recombination substantially less likely. The new vector system may improve the safety of vectors for vaccination and cancer therapy and may also provide safer backbones for further vector development, such as helper-dependent and hybrid vectors.     

Adenovirus as an integrating vector

Recombinant adenoviral vectors have served as one of the most efficient gene delivery vehicles in vivo thus far. Multiply attenuated or completely gutless adenoviral vectors have been developed to achieve long-term gene expression in animal models by overcoming cellular immunity against de novo synthesized adenoviral proteins. However, since adenovirus lacks native integration machinery, the goal of gene therapy obtaining permanent expression cannot be realized with current adenoviral vector systems. Recent studies have shown that replication-incompetent adenoviral vectors randomly integrate into host chromosomes at frequencies of 0.001-1% of infected cells. To improve the integration frequencies of adenoviral vectors, a variety of hybrid vectors combining the highly efficient DNA delivery of adenovirus with the integrating machinery of retroviruses, adeno-associated viruses, and transposons, have been emerging. These hybrid vectors have shown promise, at least in in vitro systems. Furthermore, adenoviral vectors have shown potential as gene targeting vectors. These developments should eventually lead to more effective gene therapy vectors that can transduce a myriad of cell types stably in vivo.     

Development of a suspension serum-free helper-dependent adenovirus production system and assessment of co-infection conditions

Helper-dependent adenovirus (HDAd), deleted in all viral protein-coding sequences has been designed to reduce immune response and favor long-term expression of therapeutic genes in clinical programs. Its production requires co-infection of E1-complementing cells with helper adenovirus (HAd). Significant progresses have been made in the molecular design of HDAd, but large scale production remains a challenge. In this work, a scalable system for HDAd production is designed and evaluated focusing on the co-infection step. A human embryo kidney 293 (293) derived cell line, the 293SF/FLPe was generated to produce efficiently HDAd while restricting the packaging of HAd. This cell line was adapted to grow in suspension and in serum-free medium. Multiplicity of infection (MOI) of HDAd ranging from 0.1 to 50 was evaluated in presence of HAd at a MOI of 5. Optimal MOIs for HDAd amplification were found in the range of 5-10. HAd contamination was only 1%. These results were validated in a 3 L bioreactor under controlled operating conditions where a higher HDAd yield of 2.6 x 10(9) viral particles (VP)/mL or 3.5 x 10(8) infectious units (IU)/mL of HDAd was obtained.     

Neuronal gene transfer by baculovirus-derived vectors accommodating a neurone-specific promoter

Recombinant baculoviruses have been employed as gene delivery vectors for mammalian cells, including neurones, during recent years. The aim of the current study was to develop a new recombinant baculovirus vector capable of enhancing gene expression in neurones. A hybrid promoter constructed by fusing the enhancer of human cytomegalovirus (CMV) immediately early promoter to the human platelet-derived growth factor (PDGF) beta-chain promoter was placed into a baculovirus expression cassette. In cultured neurones, baculovirus vectors containing the hybrid promoter augmented transgene expression up to 100-fold greater than that mediated by titre-matched baculovirus vectors with the PDGF promoter alone. Double immunostaining of tissue sections collected from the striatum and the retina injected with the new baculovirus vector demonstrated its specificity in driving gene expression almost exclusively in neurones, confirming the feasibility of using a tissue-specific promoter in the context of baculovirus vectors to provide cell type-specific transgene expression. The attributes of the new baculovirus vector might have practical implications for gene therapy in the nervous system.     

Strain-specific rate of shutdown of CMV enhancer activity in murine liver confirmed by use of persistent [E1(-), E2b(-)] adenoviral vectors

The systemic delivery of [E1(-)] adenoviral (Ad) vectors encoding a transgene results in efficient viral uptake and abundant transgene expression in the liver. However, [E1(-)]Ad vector persistence is transient due to cytotoxic T lymphocyte (CTL)-mediated loss of the Ad-infected cells. Our laboratory has previously demonstrated that additional modifications to the [E1(-)]Ad vector genome, by deletion of the Ad E2b genes, significantly decreased virus-genome-derived gene expression and simultaneously improved the long-term performance of the resultant [E1(-), E2b(-)]Ad vector. In this study, we confirmed that [E1(-), E2b(-)]Ad vector genomes could persist equally well in C57Bl/6 or Balb/c mouse hepatocytes. Despite vector genome persistence, we observed a strain-dependent variability in the duration of CMV enhancer/promoter-driven transgene expression in the liver. While Balb/c mice rapidly shut down [E1(-), E2b(-)]Ad-derived transgene expression, C57Bl/6 mice allowed for prolonged transgene expression. This occurred even when both strains were crossed into a severe combined immune-deficient background, demonstrating that host adaptive immune responses are not responsible for the phenomenon. Furthermore, differential methylation of the CMV enhancer/promoter was also not demonstrated in either strain of mouse, eliminating this mechanism as causative. Thus, alternative mechanisms for this phenomenon are discussed.     

Chimeric herpes simplex virus/adeno-associated virus amplicon vectors

Chimeric or hybrid herpes simplex virus type 1/adeno-associated virus amplicon vectors combine the large transgene capacity of HSV-1 with the potential for site-specific genomic integration and stable transgene expression of AAV. These chimeric vectors have been demonstrated to support transgene expression for significantly longer periods than standard HSV-1 amplicons. Moreover, HSV/AAV hybrid vectors can mediate integration at the AAVS1 pre-integration site on human chromosome 19 at a relatively high rate, although random integration has also been observed. One major remaining hurdle of HSV/AAV hybrid vectors is the low packaging efficiency and titers when AAV rep sequences are included in the amplicon vector. In the conditions prevalent during the replication/packaging of HSV/AAV hybrid amplicons into HSV-1 virions, in particular the presence of HSV-1 replication factors and AAV Rep protein, at least three different viral origins of DNA replication are active: the HSV-1 ori, the AAV inverted terminal repeats (ITRs), and the p5 promoter/ori driving expression of the AAV rep gene. A detailed understanding of the properties of these origins of DNA replication and the molecular mechanisms of interactions between them, may allow designing novel hybrid vectors that allow the efficient and precise integration of large transgenes in the human genome.     

Comparative analysis of vector biodistribution, persistence and gene expression following intravenous delivery of bovine, porcine and human adenoviral vectors in a mouse model

Nonhuman adenoviruses including bovine adenovirus serotype 3 (BAd3) and porcine adenovirus serotype 3 (PAd3) can circumvent pre-existing immunity against human adenovirus serotype 5 (HAd5) and are being developed as alternative vectors for gene delivery. To assess the usefulness of these vectors for in vivo gene delivery, we compared biodistribution, persistence, state of vector genome, and transgene and vector gene expression by replication-defective BAd3 and PAd3 vectors with those of HAd5 vector in a FVB/n mouse model following intravenous inoculation. BAd3 vector efficiently transduced the heart, kidney and lung in addition to the liver and spleen and persisted for a longer duration compared to PAd3 or HAd5 vectors. Biodistribution of PAd3 vector was comparable to that of HAd5 vector but showed more rapid vector clearance. Only linear episomal forms of BAd3, PAd3, and HAd5 vector genomes were detected. All three vectors efficiently expressed the green fluorescent protein (GFP) transgene proportionate to the vector genome copy number in various tissues. Furthermore, leaky expression of vector genes, both the early (E4) and the late (hexon) was observed in all three vectors and gradually declined with time. These results suggest that BAd3 and PAd3 vectors could serve as an alternative or supplement to HAd5 for gene delivery applications.     

Immune responses to lentiviral vectors

Efficient delivery and sustained expression of a therapeutic gene into human tissues are the requisite to accomplish the high expectations of gene therapy. A major challenge has concerned development of gene transfer systems capable of efficient cell transduction and transgene expression without harm to the recipient. A lot of work has been done to demonstrate the efficacy of gene therapy in animal models that mimic situations in humans. Use of lentiviral vectors (LVs) offers multiple advantages for gene replacement therapy, because they combine efficient delivery, ability to transduce proliferating and resting cells, capacity to integrate into the host chromatin to provide stable long-term expression of the transgene, absence of any viral genes in the vector and absence of interference from preexisting viral immunity. However, one of the major barriers to stable gene transfer by LVs and other viral vectors is the development of innate and adaptive immune responses to the delivery vector and the transferred therapeutic transgene. Since this greatly hinders the therapeutical benefits of gene therapy by LVs, developing strategies to overcome the host immune response to the transfer vector and the transgene is a matter of current investigation.     

Gene therapy with helper-dependent adenoviral vectors: lessons from studies in large animal models

Helper-dependent adenoviral vectors (HDAd) are deleted of all viral genes and they can efficiently transduce a wide variety of dividing and non-dividing cells to mediate high transgene expression levels. Unlike early generation adenoviral vectors, the absence of viral genes in HDAd results in long-term transgene expression without chronic toxicity and permits a large cloning capacity of 36 kb. Moreover, HDAd genomes exist extra-chromosomally thus minimizing the risks of germline transmission and insertional mutagenesis. For these reasons, HDAd offers tremendous potential for in vivo gene therapy. This chapter reviews preclinical studies using HDAd in large animal models to assess safety and efficacy in a wide variety of gene therapy applications.