Adenoviral vectors for gene replacement therapy

Adenovirus-based vectors are promising vehicles for gene replacement therapy due to their ability to efficiently transduce a wide variety of proliferating and non-proliferating cells. Over the past decade, different versions of adenoviral vectors (Ads) have been developed. These vectors can be classified into two major categories, based on whether the viral coding sequences are partially (first or second-generation Ads) or completely deleted (helper-dependent or gutted Ads). Both types of Ads have been tested in a variety of gene delivery studies, and major obstacles to their clinical application have been identified. Currently, innate and adaptive host immune responses to Ads remain major challenges, limiting both the initial viral dose and the effectiveness of subsequent administrations. Recent developments in vector design and delivery methods have improved the potential of Ads for successful gene therapy application.     

Adenoviral vectors for mucosal vaccination against infectious diseases

Adenoviral vector has been extensively studied as a vaccine platform because of its ability to induce potent cellular and humoral immunity. One main advantage of adenoviral vectors is their natural tropism for mucosal surfaces, which makes them ideal for the purpose of mucosal vaccination against pathogens that preferentially initiate infection at the mucosal site. The current understanding of mucosal immunity suggests that mucosal vaccination is far superior to parenteral vaccination in protecting mucosal surfaces. Mucosal vaccination is particularly relevant to those infections for which parenteral immunization strategies have failed to confer protection. This review examines the use of adenoviral vector at mucosal sites for infectious disease against which the current vaccination strategies have been unsuccessful in eliciting protection. Data from animal models have suggested that adenoviral vectors are effective in protecting against infections caused by HIV, herpes simplex virus and Mycobacterium tuberculosis. We believe that these encouraging results will lead to further evaluation in clinical trials in the near future.     

RhoA基因shRNA表达载体的构建及鉴定

目的:构建针对RhoA基因的,hRNA表达载体并进行鉴定.方法:针对人RhoA的mRNA序列,设计并合成编码的shRNA的两条寡核苷酸序列,经退火成互补双链,再克隆至pGPU6/GFP/Neo质粒,构建重组体pGPU6/GFP/NeoRhoA,进行酶切及测序鉴定,然后脂质体转染LoVo细胞.荧光显微镜观察绿色荧光蛋白的表达.结果:将合成的DNA序列退火后克隆到载体,酶切及测序证实质粒为所需的序列.结论:成功地构建了针对RhoA基因的shRNA表达载体,为下一步进行RNAi的相关研究奠定了基础.     

CDK1靶向shRNA质粒载体的构建及鉴定

目的:设计并构建CDKI -shRNA质粒表达载体,转染肝癌HepG2细胞,验证shRNA对肝癌HepG2细胞CDK1基因的沉默效应,便于进一步研究CDK1的功能.方法:设计针对CDK1 mRNA靶序列的小发夹状RNA,化学合成含茎环结构的正义链与反义链,退火形成带内切酶粘/平末端的双链后,与酶切后的pSIREN-RetroQ-ZsGreen载体片段进行连接、转化.经测序等方法鉴定重组克隆.将重组载体质粒经脂质体包裹转染肝癌HePG2细胞,realtime PCR及Western blot检测RNA干扰效果.结果:重组克隆经测序证实插入的序列正确,realtime PCR及Western blot检测证实设计的3条RNA干扰序列有效沉默了肝癌HepG2细胞中的内源性CDKI.结论:所制备的CDK1-shRNA在肝癌HepG2细胞中可获得高效转染,并能产生特异性的基因沉默效应,以CDK1为靶向的shRNA能够有效下调CDK1基因的表达,对周期依赖激酶CDK1功能的研究奠定基础.     

Production of genetically modified cells expressing specific transgenes by retroviral vectors for gene therapy

Summary Techniques and protocols are described for the generation of genetically modified cells that can be used for gene therapy. Primary fibroblast cultures are established from skin biopsies, maintained in culture, frozen for long-term storage, and retrieved when necessary. Retroviral packaging cell lines are generated by transfection of DNA into retroviral packaging cells by calcium-phosphate precipitation method or by lipofection method. To generate cell lines expressing high titer virus, individual colonies of cells are cloned and the virus titer is determined. Virus collected from packaging cells expressing high titer virus is then used to infect primary fibroblasts. To obtain fibroblast cell lines expressing high amounts of transgenes, individual cells can be cloned to generate clonal cell lines. Although the methods described here are for fibroblasts, the same methods or modification of the methods can be used for other cell types.     

Adenoviral vector-mediated gene transfer for human gene therapy

Human gene therapy promises to change the practice of medicine by treating the causes of disease rather than the symptoms. Since the first clinical trial made its debut ten years ago, there are over 400 approved protocols in the United States alone, most of which have failed to show convincing data of clinical efficacy. This setback is largely due to the lack of efficient and adequate gene transfer vehicles. With the recent progress in elucidating the molecular mechanisms of human diseases and the imminent arrival of the post genomic era, there are increasing numbers of therapeutic genes or targets that are available for gene therapy. Therefore, the urgency and need for efficacious gene therapies are greater than ever. Clearly, the current fundamental obstacle is to develop delivery vectors that exhibit high efficacy and specificity of gene transfer. Recombinant adenoviruses have provided a versatile system for gene expression studies and therapeutic applications. Of late, there has been a remarkable increase in adenoviral vector-based clinical trials. Recent endeavors in the development of recombinant adenoviral vectors have focused on modification of virus tropism, accommodation of larger genes, increase in stability and control of transgene expression, and down-modulation of host immune responses. These modifications and continued improvements in adenoviral vectors will provide a great opportunity for human gene therapy to live up to its enormous potential in the second decade.     

Adenoviral gene delivery for HIV-1 vaccination

The AIDS epidemic continues to spread throughout nations of Africa and Asia and is by now threatening to undermine the already frail infrastructure of developing countries in Sub-Saharan Africa that are hit the hardest. The only option to stem this epidemic is through inexpensive and efficacious vaccines that prevent or at least blunt HIV-1 infections. Despite decades of pre-clinical and clinical research such vaccines remain elusive. Most anti-viral vaccines act by inducing protective levels of virus-neutralizing antibodies. The envelope protein of HIV-1, the sole target of neutralizing antibodies, is constantly changing due to mutations, B cell epitopes are masked by heavy glycosylation and the protein's structural unfolding upon binding to its CD4 receptor and chemokine co-receptors. Efforts to induce broadly cross-reactive virus-neutralizing antibodies able to induce sterilizing or near sterilizing immunity to HIV-1 have thus failed. Studies have indicated that cell-mediated immune responses and in particular CD8+ T cell responses to internal viral proteins may control HIV-1 infections without necessarily preventing them. Adenoviral vectors expressing antigens of HIV-1 are eminently suited to stimulate potent CD8+ T cell responses against transgene products, such as antigens of HIV-1. They performed well in pre-clinical studies in rodents and nonhuman primates and are currently in human clinical trials. This review summarizes the published literature on adenoviral vectors as vaccine carriers for HIV-1 and discusses advantages and disadvantages of this vaccine modality.     

Gateway compatible vectors for analysis of gene function in the zebrafish.

The recent establishment of recombination-based cloning systems has greatly facilitated the analysis of gene function by allowing rapid and high-efficiency generation of plasmid constructs. However, the use of such an approach in zebrafish requires the availability of recombination-compatible plasmids that are appropriate for functional studies in zebrafish embryos. In this work, we describe the construction and validation of Gateway compatible vectors based on commonly used zebrafish plasmids. We have generated pCS-based plasmids that allow rapid generation of both N-terminal and C-terminal fusion proteins, and we demonstrate that mRNA synthesized from these plasmids encodes functional native or fusion proteins in injected zebrafish embryos. In parallel, we have established similar Gateway plasmids containing Tol2 cis elements that promote efficient integration into the zebrafish genome and allow expression of native or fusion proteins in a tissue-specific manner in the zebrafish embryo. Finally, we demonstrate the use of this system to rapidly identify tissue-specific cis elements to aid the establishment of blood vessel-specific transgenic constructs. Taken together, this work provides an important platform for the rapid functional analyses of open reading frames in zebrafish embryos.     

Adenoviral vectors coated with cationic PEG derivatives for intravaginal vaccination against HIV-1

Mucus layer coating the vaginal epithelium represents a barrier for intravaginally delivered recombined adenoviral (rAd) vectors, but it could be overcome by proper polyethylene glycol (PEG) modification. Here we synthesized two cationic PEG derivatives, amino-(EO)_n/(AGE)_m-Cyss (APCs). The polymers contained neutral linear PEG (2–5 kDa) to provide a hydrophilic surface and amine pendants to provide positive charge for coating negatively charged rAd by physical adsorption. Given proper molecular composition, the polymer (5k-APC) could coat rAd without causing aggregation, facilitating its mucus penetrating ability and enhancing gene expression both in vitro and in vivo. With HIVgag as the model antigen, the polymer-rAd complexes were administered intravaginally to elicit both systemic and mucosal immune responses. 5k-APC-rAd immunization elicited robust HIVgag-specific cellular responses and also induced higher antigen-specific serum IgG. More importantly, mice immunized with 5k-APC-rAd showed higher level of IgA in vaginal lavage fluid. These findings suggest that 5k-APC-rAd is a promising system for intravaginal immunization against infectious diseases such as HIV within the vaginal tract.     

靶向HBVs和e抗原基因的shRNA表达载体共转染对HBV抗原表达的抑制作用

目的:探索靶向乙型肝炎病毒HBsAg基因的shR-NA表达载体Pgs1、Pgs2、Pgs3及靶向HBeAg基因shRNA表达载体psiHBV4、psiHBV5、psiHBV6共转染,对体外培养HepG2.2.15细胞中的HBV抗原表达的抑制作用。方法:以质粒PTZ为阴性对照,将自行构建的靶向HBVs和e抗原基因的shRNA表达载体按不同组合共转染HepG-2.2.15细胞;继续培养24 h后用MEIA分别检测细胞裂解液和培养上清中HBsAg和HBeAg的表达水平。结果:psiHBV4+PgS2、psiH-BV4+PgS3在联合转染时,在细胞上清和裂解液中对HBsAg和HBeAg表达都有显著抑制作用(P0.05),psiHBV5+PgS1、psiHBV6+PgS3对裂解液HBeAg表达抑制作用不显著(P0.05)。结论:靶向HBs和HBe基因的两种载体psiH-BV4+PgS2、psiHBV4+PgS3共转染比单个载体转染更能显著减少HBV抗原的表达。     

In vivo post-transcriptional gene silencing of alpha-1 antitrypsin by adeno-associated virus vectors expressing siRNA

alpha-1 Antitrypsin (AAT) deficiency is one of the most common genetic diseases in North America, with a carrier frequency of approximately 4% in the US population. Homozygosity for the most common mutation (Glu342Lys, PI(*)Z) leads to the synthesis of a mutant protein, which accumulates and polymerizes within hepatocytes rather than being efficiently secreted. This lack of secretion causes severe serum deficiency predisposing to chronic lung disease. Twelve to fifteen percent of patients with PI(*)ZZ also develop liver disease, which can be severe, even in infancy. This is thought to be due to toxic effects of the accumulated mutant Z-AAT within the hepatocyte. Thus, an approach to reduce AAT-deficient liver disease will likely require some mechanism to decrease the amount of Z-AAT within hepatocytes. In this report, we describe studies of small-interfering RNAs (siRNAs) designed to downregulate endogenous AAT within hepatocytes. Three different siRNA sequences were identified and cloned into a recombinant adeno-associated virus (rAAV) backbone, either singly or as a trifunctional (3X) construct. Each had activity independently, but the levels of AAT expression in cell culture models showed the greatest decrease with the 3X construct, resulting in levels that were five-fold lower than controls. The rAAV-3X-siRNA was then packaged into AAV8 capsids and used in vivo to transduce the livers of human Z-AAT overexpressing transgenic mice. Those studies showed a decrease in total human AAT, a clearing of Z-AAT accumulation by immunohistochemistry, and a decrease in monomer Z-AAT within the liver within 3 weeks after vector injection. The rAAV8-3X-siRNA vector may hold promise as a potential therapy for patients with AAT liver disease.     

Episomal vectors for gene therapy

The increasing knowledge of the molecular and genetic background of many different human diseases has led to the vision that genetic engineering might be used one day for their phenotypic correction. The main goal of gene therapy is to treat loss-of-function genetic disorders by delivering correcting therapeutic DNA sequences into the nucleus of a cell, allowing its long-term expression at physiologically relevant levels. Manifold different vector systems for the therapeutic gene delivery have been described over the recent years. They all have their individual advantages but also their individual limitations and must be judged on a careful risk/benefit analysis. Integrating vector systems can deliver genetic material to a target cell with high efficiency enabling long-term expression of an encoded transgene. The main disadvantage of integrating vector systems, however, is their potential risk of causing insertional mutagenesis. Episomal vector systems have the potential to avoid these undesired side effects, since they behave as separate extrachromosomal elements in the nucleus of a target cell. Within this article we present a comprehensive survey of currently available episomal vector systems for the genetic modification of mammalian cells. We will discuss their advantages and disadvantages and their applications in the context of basic research, biotechnology and gene therapy.     

Th2 T cells expressing transgene PDGF-A serve as vectors for gene therapy in autoimmune demyelinating disease.

We hypothesized that T cells can be genetically modified to express growth factor transgene products capable of inducing oligodendrocyte progenitor proliferation. Autoreactive T cells isolated from SWXJ mice immunized with the p139-151 determinant of myelin proteolipid protein (PLP) were transfected with an antigen-inducible transgene for platelet-derived growth factor-A (PDGF), a growth factor important in regulating the development of oligodendrocytes. Isolated antigen-specific T cell clones expressed the PDGF transgene when stimulated with PLP 139-151 peptide and produced biologically active PDGF capable of inducing proliferation of oligodendrocyte progenitor cells. Furthermore, upon adoptive transfer, the PDGF transfected T cells migrated to the CNS and ameliorated ongoing disease. Our data indicate that autoreactive memory Th2 cells can be genetically modified so that upon engagement with self antigen they produce regenerative growth factors capable of mediating tissue repair during autoimmune disease.     

Alphavirus vectors for gene therapy applications

High-titer alphavirus vectors have been generated for efficient gene delivery both in vitro and in vivo. Studies on CNS infection via intranasal and peripheral injections with virulent and avirulent replication-competent Semliki Forest virus (SFV) strains has demonstrated the potential of gene delivery. Replication-deficient alphavirus particles have shown high local transgene expression of a transient nature in rodent brain. Alphavirus vectors have been demonstrated to induce apoptosis in infected human tumor cell lines and SFV vectors expressing interleukin-12 resulted in tumor regression in a B16 murine melanoma model. Repeated SFV injections led to stronger anti-tumor effects without immunogenic response detected against SFV. It has also been shown that intra-tumoral SFV-injections into nude mice with implanted human lung carcinomas led to tumor regression. Likewise, injection of replicative SFV-LacZ RNA resulted in tumor response as well as prophylactic protection against tumor formation. Alphaviruses have also showed potential in vaccine production. Additionally, modifications in the envelope structure of Sindbis virus resulted in substantial change in host range and demonstrated the feasibility of targeting alphavirus vectors. Moreover, SFV has been used as an expression vector for the generation of high-titer retrovirus-like particles. Recent alphavirus vector development has introduced novel non-cytopathogenic vectors, tightly temperature-regulated vectors as well as replication-persistent forms that should prolong the duration of expression. Alphavirus vectors can therefore be considered as highly potential gene delivery vehicles for future gene therapy applications, especially where only short-term expression is required, or even preferred.     

Nonviral vectors for cancer gene therapy: prospects for integrating vectors and combination therapies

Gene therapy has the potential to improve the clinical outcome of many cancers by transferring therapeutic genes into tumor cells or normal host tissue. Gene transfer into tumor cells or tumor-associated stroma is being employed to induce tumor cell death, stimulate anti-tumor immune response, inhibit angiogenesis, and control tumor cell growth. Viral vectors have been used to achieve this proof of principle in animal models and, in select cases, in human clinical trials. Nevertheless, there has been considerable interest in developing nonviral vectors for cancer gene therapy. Nonviral vectors are simpler, more amenable to large-scale manufacture, and potentially safer for clinical use. Nonviral vectors were once limited by low gene transfer efficiency and transient or steadily declining gene expression. However, recent improvements in plasmid-based vectors and delivery methods are showing promise in circumventing these obstacles. This article reviews the current status of nonviral cancer gene therapy, with an emphasis on combination strategies, long-term gene transfer using transposons and bacteriophage integrases, and future directions.     

Layer-by-layer self-assembly vectors for gene delivery

The success of gene therapy largely relies on the development of high-efficient and low-toxic gene delivery vectors. Nanovector-based delivery of nucleic acids is a very promising approach for the effective transfer of genetic materials into cells. Compared with encapsulating of nucleic acids inside biodegradable nanoparticles which often suffers from low encapsulation efficiency and degradation of the loaded therapeutic gene, the layer-by-layer self-assembly vectors prepared by the surface adsorption of gene/polycation multilayered films on colloidal particles using layer-by-layer technique are a potent gene delivery system in offering efficient loading of nucleic acids, controlling the release of the loaded gene in physiological environment and targeting to a particular site or a specific cell type in the body. This review focuses on the preparation, advantages, application and the probable associated drawbacks of layer-by-layer self-assembly vectors for gene delivery.