Gene transfer strategies for the physiologist

Foreign genes can be introduced into whole animals using methods of germline transgenesis and somatic gene delivery. While germline transgenesis can generate useful animal models for genetic studies, it can be costly, time-consuming and requires the use of a large number of animals. An alternative means of gene transfer is to deliver genes to somatic cells using non-viral and viral technologies. Non-viral methods such as naked DNA injection, electroporation and liposome/cation lipid-mediated gene transfer are relatively inefficient. In contrast, viruses are effective vehicles that carry foreign genes into a cell rapidly and efficiently. Here we illustrate the usefulness of adenoviral vectors to express a potent and specific inhibitor of cAMP-dependent protein kinase (PKA) to study the role of cyclic 3',5'-cyclic AMP (cAMP) in the osmotic regulation of the vasopressin gene in a transgenic rat model. The ability to modify endogenous systems within specific cells in a whole animal model allows gene effects to be studied with physiological relevance. The combination of molecular biology and integrative physiology is a powerful application that can aid in the elucidation of how gene function can translate into complex systems in an organism.     

Gene copy number variation and common human disease

Fanciulli M, Petretto E, Aitman TJ. Gene copy number variation and common human disease. Variation in gene copy number is increasingly recognized as a common, heritable source of inter‐individual differences in genomic sequence. The role of copy number variation is well established in the pathogenesis of rare genomic disorders. More recently, germline and somatic copy number variation have been shown to be important pathogenic factors in a range of common diseases, including infectious, autoimmune and neuropsychiatric diseases and cancer. In this review, we describe the range of methods available for measuring copy number variants (CNVs) in individuals and populations, including the limitations of presently available assays, and highlight some key examples of common diseases in which CNVs have been shown clearly to have a pathogenic role. Although there has been major progress in this field in the last 5 years, understanding the full contribution of CNVs to the genetic basis of common diseases will require further studies, with more accurate CNV assays and larger cohorts than have presently been completed.     

Gene therapy: how to target the kidney. Promises and pitfalls

The success of gene therapy strongly depends on an efficient delivery system to allow local transfer and expression of the therapeutic gene in the target organ or tissue. Vector systems have been improved and many show promise. There are two different categories of delivery vehicles: non-viral and viral vectors, both with advantages and disadvantages that must be taken into consideration in view of the final aim. Compared to other solid organs, the kidney offers the main advantage of access by different routes that dictate different sites of transfection. Thus, the choice of the delivery vehicle and administration route has to take account which cells are to be specifically targeted by the gene transfer approach. This concept will be discussed in the first part of the review. Using a gene therapy approach, improvements of renal function and interstitial inflammation have been achieved in experimental models of glomerulonephritis and tubulo-interstitial damage. Gene therapy applied to renal transplantation has shown promising results in rodents, almost controlling acute rejection. Finally, the development of animal models resembling the clinical features of human genetic renal disorders offers a first step towards new treatments among which gene therapy could become reality in the near future. The main findings concerning the suitability of gene therapy for slowing the progression of kidney diseases, and preventing acute renal graft rejection, or treating genetic disorders, are discussed.     

逆转录病毒介导的基因治疗中的安全性检测

逆转录病毒介导的基因治疗中安全性的最大危险是产生有复制能力的逆转录病毒（RCR）。本实验对经3T3扩增的样品进行 S~+L~-分析、标记拯救分析和PCR扩增方法检测RCR。比较三种方法,标记拯救分析比S~+L~-分析结果容易判断,且PCR可以从10~5个无病毒基因的细胞中检测出一个带有病毒基因的细胞。3T3扩增可提高灵敏度约10倍。安全性是基因治疗首要考虑的问题,本方法为其提供了保证。     

Effects of Cytokine Gene Therapy on Particulate-Induced Inflammation in the Murine Air Pouch

Retroviral vectors encoding the human IL-1 antagonist (IL-1Ra) gene and the human tumor necrosis factor soluble receptor (sTNF-R) gene were investigated using an in vivo model of the inflammatory response to orthopedic wear debris. Air pouches established in BALB/c mice were injected with polymethylmethacrylate (PMMA) particles to provoke an inflammatory reaction, and infected with retroviral vectors expressing IL-1Ra, sTNF-R or a LacZ marker gene. Pouch membranes and fluids were harvested after 48 or 72 hours for analyses. Positive PCR reactions for Neo genes were observed specifically in DNA extracted from the membrane of retroviral-infected pouches. ELISA assays revealed the presence of human IL-1Ra in pouch fluid from DFG-IRAP-Neo transduced mice, but not control animals. Histological evaluation indicated that the IL-1Ra gene transfer was associated with markedly decreased inflammation in the model, with resolution of the edematous phase of the reaction, decreased pouch fluid accumulation, and lowered macrophage influx. The data suggest that the air pouch model represents a useful tool to evaluate gene therapy, and demonstrate that IL-1Ra gene therapy may be an appropriate therapeutic approach to inflammation.     

Gene therapy strategies to prevent autoimmune disorders

Autoimmunity accounts for a significant percentage of human disease and remains a challenging syndrome to treat. While systemic immunosuppression can be beneficial, the associated toxicity of the pharmacologic agents necessitates an antigen-specific approach to silence, eradicate or prevent the genesis of autoreactive immune cells. Gene therapy offers the possibility of providing precise antigen-targeted therapies, thereby sparing the patient the significant toxicity associated with lifelong commitment to chemical immunosuppressives. Gene-based therapies could include, but are not limited to the manipulation of immune networks of tolerance by antigen presenting cell engineering, pro-inflammatory cytokine blockade using soluble antagonists expressed from viral vectors as well as modulation of immune regulatory networks. The potential utility of gene therapy strategies promoting tolerance in two model autoimmune disorders, type I diabetes mellitus and rheumatoid arthritis are discussed in this review.     

Pathway‐Guided Identification of Gene‐Gene Interactions

Assessing gene‐gene interactions (GxG) at the gene level can permit examination of epistasis at biologically functional units with amplified interaction signals from marker‐marker pairs. While current gene‐based GxG methods tend to be designed for two or a few genes, for complex traits, it is often common to have a list of many candidate genes to explore GxG. We propose a regression model with pathway‐guided regularization for detecting interactions among genes. Specifically, we use the principal components to summarize the SNP‐SNP interactions between a gene pair, and use an L1 penalty that incorporates adaptive weights based on biological guidance and trait supervision to identify important main and interaction effects. Our approach aims to combine biological guidance and data adaptiveness, and yields credible findings that may be likely to shed insights in order to formulate biological hypotheses for further molecular studies. The proposed approach can be used to explore the GxG with a list of many candidate genes and is applicable even when sample size is smaller than the number of predictors studied. We evaluate the utility of the proposed method using simulation and real data analysis. The results suggest improved performance over methods not utilizing pathway and trait guidance.     

Envelope and Long Terminal Repeat Sequences of an Infectious Murine Leukemia Virus from a Human SCLC Cell Line: Implications for Gene Transfer

Development of methods for gene transfer into specific cell types or tissues is important for experimental research as well as clinical therapeutical approaches. We report here the cloning and characterization of the envelope (env) gene and the U3 region of a retrovirus from an infected human Small Cell Lung Cancer (SCLC) cell line. The replication of this murine retrovirus is also fully supported by other lung cancer cell lines of different histological origin. We present evidence that a long terminal repeat (LTR)-β-galactosidase (β-Gal) reporter construct performed as well as an analogous cytomegalovirus (CMV) promoter β-Gal construct in the human lung epithelial cell line A549 and in the human larynx carcinoma cell line HEp2. This revised version was published online in July 2006 with corrections to the Cover Date.     

Gene therapy of scarring: a lesson learned from fetal scarless wound healing.

Cutaneous wounding in adult humans and higher vertebrate animals results in scar formation. In contrast, both human and animal fetuses, at early gestational ages, exhibit skin wound healing without scarring. This distinction suggests that the repair of adult wounds by skin regeneration, rather than by fibrosis, may be achieved if adult wounds can be modified to mimic the healing process of fetal wounds. The development of gene therapy offers the possibility to specifically enhance or block the gene expression of cytokines and extracellular molecules, and thus convert adult wound healing into a healing process more similar to tissue regeneration. This article reviews the characteristics of fetal wound repair focusing on cytokine profiles and the inflammatory response to dermal injury. Also included are new developments in gene transfer techniques as well as their application in wound healing. Finally, the authors propose possible strategies of wound gene therapy, to reduce wound scarring and to promote tissue regeneration.     

Hepatitis B Virus Vector Carries a Foreign Gene into Liver Cells In Vitro

Hepatitis B viruses (HBV) specifically target the liver, where they efficiently infect quiescent hepatocytes. Thus, HBV virus has potential to be used as vectors for liver-directed gene transfer. We constructed a new HBV-based vector system. It is composed of transfer vector for transferring a foreign gene, green fluorescence protein (GFP) gene, and a helper vector. When the transfer vector and the helper vector were cotransfected into HepG2 cells, the recombinant HBV (rHBV) particles were generated by trans-complementation between two vectors. The rHBV particles carrying the foreign gene were identified by the Southern blot assay. To test gene delivery and the transduction of the rHBV, we infected primary human hepatocytes and immortalized, HepG2 cells with rHBV in vitro. The results using fluorescence microscopy confirmed that the inserted GFP gene was successfully transferred and expressed both in primary human hepatocytes and HepG2 cells.     

PLUNC基因启动子区荧光素酶报告载体的构建与鉴定

目的构建人PLUNC（palate,lung and nasal epithelium clone）基因启动子区C-1888T SNP位点不同单倍型荧光素酶报告基因表达载体。方法利用PCR技术得到1888位点不同基因型的PLUNC基因启动子区目的片段．将其分别插入到pMD18-T载体和pGL3-enhancer荧光素酶报告基因载体中并测序。结果成功构建了人类PLUNC基因C-1888T SNP位点上的2种不同纯合子基因型（CC型和TT型）的荧光素酶报告载体（pGL3-enhancer—T型和pGL3-enhancer—C型）,且测序得以证实。结论成功构建出的荧光素酶报告载体,为研究PLUNC基因不同的1888SNF位点能否调控不同的PLUNC基因表达提供了基本实验条件。     

Gene delivery in bone tissue engineering: progress and prospects using viral and nonviral strategies

Bone tissue loss as a consequence of the natural aging process or as a result of trauma and degenerative disease has led to the need for procedures to generate cartilage and bone for a variety of orthopedic applications. The ability to transfer genes into multipotential mesenchymal stem cells, while still in its infancy, offers considerable therapeutic hope in a variety of musculoskeletal disorders. However, the choice of gene delivery method is key. This review examines the various techniques and methods currently available to enable gene transfer into a target population from viral methods (transduction) to nonviral (transfection) methods and the limitations associated with each method. The potential applications and current understanding of each method are presented. Given the demographic challenge of an aging population, the ultimate goal remains the development of simple, safe, and reproducible strategies for gene delivery that will address the pressing orthopedic clinical imperatives of many.     

EcoRI Restriction Fragment-Length Polymorphism of the Human Immunoglobulin Variable Lambda 8 (IGLV8) Subgroup Reveals a Gene Family

The human immunoglobulin lambda locus (IGL) maps on chromosome 22q11.1–q11.2 and directs the synthesis of lambda-type Ig light chains. This locus is formed by three gene clusters (VA, VB and VC) that encompass the variable coding genes and the J-C cluster plus the joining segments and the constant genes. Recently the variable lambda gene clusters were mapped by the contig methodology which located all the known functional v-lambda genes and pseudogenes. The 30 functional v-lambda genes described so far were subgrouped into ten families (VλI to VλX), but RFLP studies have estimated that the germline repertoire contains about 70 genes. Based on sequence comparisons, we defined specific oligonucleotide primers for the unique IGLV8S1 genedescribed. The cloned 244 bp product obtained from genomic DNA with these primers was sequenced and used as probe in Southern hybridization EcoRI RFLP analysis of Brazilian people. We detected the IGLV8S1 gene in a 3.7 kb EcoRI restriction fragment present in all the individuals analyzed, in agreement with the physical map of the IGL locus. Moreover, we detected an 8.0 kb EcoRI monomorphic fragment and a 6.0 kb EcoRI polymorphic fragment. These data suggest that the IGLV8 subgroup is a gene family.     

Gene expression profile class prediction using linear Bayesian classifiers

Due to recent advances in DNA microarray technology, using gene expression profiles, diagnostic category of tissue samples can be predicted with high accuracy. In this study, we discuss shortcomings of some existing gene expression profile classification methods and propose a new approach based on linear Bayesian classifiers. In our approach, we first construct gene-level linear classifiers to identify genes that provide high class-prediction accuracies, i.e., low error rates. After this screening phase, starting with the gene that offers the lowest error rate, we construct a multi-dimensional linear classifier by incorporating next best-performing genes, until the prediction error becomes minimum or 0, if possible. When we compared classification performance of our approach against prediction analysis of microarrays (PAM) and support vector machines (SVM) based approaches, we found that our method outperforms PAM and produces comparable results with SVM. In addition, we observed that the gene selection scheme of PAM could be misleading. Albeit SVM achieves relatively higher prediction performance, it has two major disadvantages: Complexity and lack of insight about important genes. Our intuitive approach offers competing performance and also an efficient means for finding important genes.     

聚乙二醇在非病毒转基因载体中的应用

非病毒转基因载体在基因治疗领域的应用日趋广泛,但目前开发的非病毒转基因载体直接体内应用由于存在诸多问题而受到限制。受药物和蛋白质聚乙二醇化后能增加目的药物和蛋白质稳定性,延长体内半衰期,降低毒副作用等效应的启发,近年有许多研究将聚乙二醇化应用于非病毒转基因载体,取得了一定的效果。     

Detection of neutralizing antibodies against human papillomaviruses (HPV) by inhibition of gene transfer mediated by HPV pseudovirions

The goal of this study was to develop a human papillomavirus (HPV) neutralization assay using HPV pseudovirions generated in vitro. For this purpose, gene transfer efficiency of HPV virus-like particles (VLPs) was improved by using direct interaction between a reporter plasmid and the VLPs. Electron microscopic observation of the interaction between DNA molecules and VLPs revealed that VLPs always interact with a single DNA molecule and that VLPs bind to the end of linearized DNA molecules. An 100-fold improvement in the gene transfer was obtained by simple interaction between a linearized DNA molecule and VLPs. Moreover, direct interaction methods offer the possibility of transferring plasmids a size higher than that of the papillomavirus genome. The approach that we developed to generate HPV-16 and HPV-31 pseudovirions proved to be suitable for testing neutralizing antibodies in human sera both after immunization and after natural infection.     

Genetic origin of human anti-insulin antibodies

Numerous studies have characterized the specificity of human anti-insulin antibodies; however, little is known about their genetic origin. To initiate molecular studies, B cells that bind human insulin were selected from the peripheral blood of diabetic donors and transformed with Epstein-Barr virus. The resulting anti-insulin B-cell lines were cultured at limiting dilution and examined for immunoglobulin heavy-chain gene rearrangements on Southern gels. These studies demonstrated the clonality of the B-cell lines and showed that multiple immunoglobulin heavy chain rearrangements are present. When the heavy-chain variable region (VH) gene from one of these Epstein-Barr virus cell lines was cloned, it was found to belong to the recently identified human VHV gene family that represents less than 1% of known human VH genes. Using the polymerase chain reaction, the germline VHV gene of the donor was amplified and sequenced. The sequences showed a high level of homology (98%) between the expressed and germline VHV gene of the donor. While antibodies reactive with autologous insulin (like other autoantibodies) are not extensively mutated from their germline configuration, two replacement substitutions are present in this IgM antibody.     

Gene transfer to dendritic cells induced a protective immunity against melanoma

Lentiviral vectors have shown promises for efficient gene transfer to dividing as well as nondividing cells. In this study, we explored lentiviral vector-mediated, the entire mTRP-2 gene transfer and expression in dendritic cells （DCs）. Adoptive transfer of DCs-expressing mTRP-2 （DC-HR＇CmT2） into C57BL/6 mouse was also assessed. Dendritic cells were harvested from bone marrow and functional DCs were proved by allogeneic mixed lymphocyte reaction. Lentiviral vectors were produced by transient transfection of 293T cells. Transduction of DCs was proved by marker gene expression and PCR and RT-PCR amplification. Implantation of the transduced DCs, depletion of immune cells as well as the survival of the mice after tumour challenge were investigated. High efficiency of gene transfer into mature DCs was achieved. The high level expression of the functional antigen （TRP-2） and induction of protective immunity by adoptive transfer of TRP-2 gene modified DCs were demonstrated. In vivo study showed a complete protection of mice from further melanoma cell challenge. In comparison, only 83% of mice survived when mTRP-2 peptide-pulsed DCs were administered, suggesting the generation of specific protection. Together, these results demonstrated the usefulness of this gene transfer to DC approach for immunotherapy of cancer and indicated that using tumour associated antigens （TAAs） for gene transfer may be potentially beneficial for the therapy of melanoma.