Analyzing the number of common integration sites of viral vectors--new methods and computer programs

Vectors based on γ-retroviruses or lentiviruses have been shown to stably express therapeutical transgenes and effectively cure different hematological diseases. Molecular follow up of the insertional repertoire of gene corrected cells in patients and preclinical animal models revealed different integration preferences in the host genome including clusters of integrations in small genomic areas (CIS; common integrations sites). In the majority, these CIS were found in or near genes, with the potential to influence the clonal fate of the affected cell. To determine whether the observed degree of clustering is statistically compatible with an assumed standard model of spatial distribution of integrants, we have developed various methods and computer programs for γ-retroviral and lentiviral integration site distribution. In particular, we have devised and implemented mathematical and statistical approaches for comparing two experimental samples with different numbers of integration sites with respect to the propensity to form CIS as well as for the analysis of coincidences of integration sites obtained from different blood compartments. The programs and statistical tools described here are available as workspaces in R code and allow the fast detection of excessive clustering of integration sites from any retrovirally transduced sample and thus contribute to the assessment of potential treatment-related risks in preclinical and clinical retroviral gene therapy studies.     

Adenoviral vectors for gene therapy

Vectors based on human adenovirus serotypes 2 (Ad2) and 5 (Ad5) of species C possess a number of features that have favored their widespread employment for gene delivery both in␣vitro and in␣vivo. However, the use of recombinant Ad2- and Ad5-based vectors for gene therapy also suffers from a number of disadvantages. These vectors possess the tropism of the parental viruses, which infect all cells that possess the appropriate surface receptors, precluding the targeting of specific cell types. Conversely, some cell types that represent important targets for gene transfer express only low levels of the cellular receptors, which lead to inefficient infection. Another major disadvantage of Ad2- and Ad5-based vectors in␣vivo is the elicitation of both an innate and an acquired immune response. Considerable attention has therefore been focused on strategies to overcome these limitations, thereby permitting the full potential of adenoviral vectors to be realized.     

Using Multivalent Adenoviral Vectors for HIV Vaccination

Adenoviral vectors have been used for a variety of vaccine applications including cancer and infectious diseases. Traditionally, Ad-based vaccines are designed to express antigens through transgene expression of a given antigen. For effective vaccine development it is often necessary to express or present multiple antigens to the immune system to elicit an optimal vaccine as observed preclinically with mosaic/polyvalent HIV vaccines or malaria vaccines. Due to the wide flexibility of Ad vectors they are an ideal platform for expressing large amounts of antigen and/or polyvalent mosaic antigens. Ad vectors that display antigens on their capsid surface can elicit a robust humoral immune response, the “antigen capsid-incorporation” strategy. The adenoviral hexon protein has been utilized to display peptides in the majority of vaccine strategies involving capsid incorporation. Based on our abilities to manipulate hexon HVR2 and HVR5, we sought to manipulate HVR1 in the context of HIV antigen display for the first time ever. More importantly, peptide incorporation within HVR1 was utilized in combination with other HVRs, thus creating multivalent vectors. To date this is the first report where dual antigens are displayed within one Ad hexon particle. These vectors utilize HVR1 as an incorporation site for a seven amino acid region of the HIV glycoprotein 41, in combination with six Histidine incorporation within HVR2 or HVR5. Our study illustrates that these multivalent antigen vectors are viable and can present HIV antigen as well as His₆ within one Ad virion particle. Furthermore, mouse immunizations with these vectors demonstrate that these vectors can elicit a HIV and His₆ epitope-specific humoral immune response.     

Adenoviral gene transfer to committed hematopoietic progenitors

High-efficiency gene transfer into ex vivo expanded human hematopoietic progenitors and precursor cells by adenovirus vectorsFrey, B.M. et al. (1998)Blood 91, 2781–2792     

Adenoviral vectors for gene transfer and therapy

Due to the very efficient nuclear entry mechanism of adenovirus and its low pathogenicity for humans, adenovirus-based vectors have become gene delivery vehicles that are widely used for transduction of different cell types, especially for quiescent, differentiated cells, in basic research, in gene therapy applications, and in vaccine development. As an important basis for their use as gene medicine, adenoviral vectors can be produced in high titers, they can transduce cells in vivo with transgenes of more than 30 kb, and they do not integrate into the host cell genome. Recent advances in the development of adenoviral vectors have brought considerable progress on issues like target cell specificity and tropism modification, long-term expression of the transgene, as well as immunogenicity and toxicity in vivo, and have suggested that the different generations of non-replicative and replicative vectors available today will each suit best for certain applications.     

第三代腺病毒载体的研究进展

第三代腺病毒载体(也称为空壳载体,辅助病毒载体等)的研究已近十年,它在研究中所表现出来的特点使大多数研究者认为它是一个很有希望的理想的基因治疗转导载体。但由于生产该类载体的生产系统比较复杂,影响因素多,至今仍不能提供临床级的病毒进行研究。该文首先总结了腺病毒(载体)的性质,回顾了第三代腺病毒载体的产生和研究历程,总结了各生产系统之间的改进与不同,以及第三代腺病毒载体在基因相关疾病中的具体应用,希望对以后空壳载体的发展有所帮助。     

Chromosomal Integration of Adenoviral Vector DNA In Vivo

So far there has been no report of any clinical or preclinical evidence for chromosomal vector integration following adenovirus (Ad) vector-mediated gene transfer in vivo. We used liver gene transfer with high-capacity Ad vectors in the FAH^Δexon5 mouse model to analyze homologous and heterologous recombination events between vector and chromosomal DNA. Intravenous injection of Ad vectors either expressing a fumarylacetoacetate hydrolase (FAH) cDNA or carrying part of the FAH genomic locus resulted in liver nodules of FAH-expressing hepatocytes, demonstrating chromosomal vector integration. Analysis of junctions between vector and chromosomal DNA following heterologous recombination indicated integration of the vector genome through its termini. Heterologous recombination occurred with a median frequency of 6.72 × 10⁻⁵ per transduced hepatocyte, while homologous recombination occurred more rarely with a median frequency of 3.88 × 10⁻⁷. This study has established quantitative and qualitative data on recombination of adenoviral vector DNA with genomic DNA in vivo, contributing to a risk-benefit assessment of the biosafety of Ad vector-mediated gene transfer.Recombinant adenovirus (Ad) vectors are under clinical development for different applications, including tumor therapy, vaccination, and gene therapy. Today, the largest number of clinical gene transfer trials has been based on Ad vectors (http://www.wiley.co.uk/genmed/clinical). Several Ad vectors are in phase III clinical trials, and two products have already been approved in China. The occurrence of malignancies due to retroviral integration and oncogene activation in a clinical trial for the treatment of children with SCID-X1 (10) has pointed to the need for a thorough preclinical evaluation of potential genotoxic effects due to chromosomal integration of gene transfer vectors as an important part of the overall risk-benefit analysis. Detailed information on genotoxicity following gene transfer is available for vectors derived from viruses of the Retroviridae and Parvoviridae families (2, 20, 23, 26, 46). Between 60 and 75% of integrations of retrovirus, lentivirus, or adeno-associated virus (AAV)-based vectors take place in or close to genes.Chromosomal integration of Ad vector DNA following gene transfer in cell culture has been analyzed in only a few studies, and even less is known about Ad vector integration in vivo. Since the life cycle of wild-type adenovirus is extrachromosomal, Ad vectors are perceived to be nonintegrating vectors. However, in earlier studies it was observed that injection of hamsters with wild-type adenovirus type 12 (Ad12) resulted in tumor formation due to chromosomal integration of virus DNA and expression of the E1A/E1B oncoproteins (33). Recent in vitro studies with Ad vectors with E1 deletions have demonstrated the occurrence of vector integration following transduction of transformed cell lines and primary cells, with the frequencies of homologous and heterologous recombination being between 10⁻³ and 10⁻⁶ and between 10⁻³ and 10⁻⁵ per cell, respectively, depending on the conditions used (12, 14, 28, 36, 37, 42, 43). Since clinical gene transfer trials, including prophylactic vaccination of healthy volunteers against infectious diseases, are performed with large amounts of vector (in general, between 10¹⁰ and 10¹³ particles), it is possible that substantial integration of adenoviral vector DNA might also occur in vivo even if integration rates were low. However, so far there has been no attempt to experimentally address the issue of Ad vector integration in vivo. We used the FAH^Δexon5 mouse model (8) of tyrosinemia type I (MIM 27670) to analyze potential homologous and heterologous recombination events between Ad vector DNA and chromosomal DNA in vivo. Tyrosinemia type I is caused by the lack of fumarylacetoacetate hydrolase, an enzyme that is involved in the tyrosine degradation pathway and that converts fumarylacetoacetate into fumaric acid and acetoacetic acid in hepatocytes (38). Loss of fumarylacetoacetate hydrolase (FAH) activity in hepatocytes results in the accumulation of toxic and mutagenic metabolites in a cell-autonomous fashion, leading after birth to an acute hepatopathy and later in life to a chronic hepatopathy. Liver damage can be prevented both in humans and in FAH-deficient animals by the administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), which blocks the tyrosine degradation pathway by inhibiting 4-hydroxphenyl pyruvate dioxygenase, thereby preventing the accumulation of the toxic compounds. The murine FAH gene is located on chromosome 7, contains 14 exons, and spans 20.5 kb.The autosomal recessive FAH^Δexon5 mouse model, in which exon 5 is disrupted by the insertion of a NeoR gene (8), has been a useful system to analyze chromosomal integration of AAV, retrovirus, Sleeping Beauty transposon, and plasmid DNA in hepatocytes (13, 25, 27, 31). Similar to human tyrosinemia type I patients with spontaneous reversions of point mutations (18), FAH-expressing hepatocytes have a strong growth advantage over FAH^−/− hepatocytes, and the developing nodules, consisting of FAH-positive [FAH⁺] hepatocytes, can be easily distinguished in an environment of FAH^−/− hepatocytes. Following injection of an FAH-expressing Ad vector with the E1 deletion (30) into FAH^−/− mice, the development of FAH⁺ nodules in the livers of the experimental animals was observed, suggesting potential chromosomal integration of vector DNA. Since transgene expression from vectors with the E1 deletion is transient, in part due to viral toxicity and an immune response directed to viral proteins expressed from the vector, integration events and their characterization were not possible. We reasoned that the use of high-capacity Ad (HC-Ad) vectors (also called “helper-dependent” or “gutless” Ad vectors) (41) not expressing any viral proteins would allow reliable data on Ad vector integration in vivo to be obtained.     

人Sef基因重组腺病毒载体的构建与鉴定

构建人Sef-L和Sef-S基因的复制缺陷型重组腺病毒表达载体, 为研究Sef的功能和作用机制以及Sef的基因治疗奠定基础。通过PCR方法以hSef的表达质粒为模板扩增得到hSef的编码序列, 亚克隆到穿梭载体pAdTrack-CMV中, 经测序验证之后, 将穿梭载体使用Pme I酶切线性化, 然后与腺病毒基因组质粒pAdEasy-1共转化大肠杆菌BJ5183, 得到重组的Ad-hSef-L和Ad-hSef-S质粒, 最后将Ad-hSef-L和Ad-hSef-S质粒使用Pac I线性化, 转染到HEK293细胞中, 包装收获病毒颗粒, 免疫印迹实验鉴定表达, 荧光素酶报告实验验证其功能。成功构建了人Sef基因的复制缺陷型重组腺病毒表达载体, 获得了有功能的Ad-hSef-L和Ad-hSef-S病毒重组子。     

人Sef基因重组腺病毒载体的构建与鉴定

构建人Sef-L和Sef-S基因的复制缺陷型重组腺病毒表达载体, 为研究Sef的功能和作用机制以及Sef的基因治疗奠定基础。通过PCR方法以hSef的表达质粒为模板扩增得到hSef的编码序列, 亚克隆到穿梭载体pAdTrack-CMV中, 经测序验证之后, 将穿梭载体使用Pme I酶切线性化, 然后与腺病毒基因组质粒pAdEasy-1共转化大肠杆菌BJ5183, 得到重组的Ad-hSef-L和Ad-hSef-S质粒, 最后将Ad-hSef-L和Ad-hSef-S质粒使用Pac I线性化, 转染到HEK293细胞中, 包装收获病毒颗粒, 免疫印迹实验鉴定表达, 荧光素酶报告实验验证其功能。成功构建了人Sef基因的复制缺陷型重组腺病毒表达载体, 获得了有功能的Ad-hSef-L和Ad-hSef-S病毒重组子。     

An Adenoviral Vector Based Vaccine for Rhodococcus equi

Rhodococcus equi is a respiratory pathogen which primarily infects foals and is endemic on farms around the world with 50% mortality and 80% morbidity in affected foals. Unless detected early and treated appropriately the disease can be fatal. Currently, there is no vaccine available to prevent this disease. For decades researchers have endeavoured to develop an effective vaccine to no avail. In this study a novel human adenoviral vector vaccine for R. equi was developed and tested in the mouse model. This vaccine generated a strong antibody and cytokine response and clearance of R. equi was demonstrated following challenge. These results show that this vaccine could potentially be developed further for use as a vaccine to prevent R. equi disease in foals.     

Adenoviral gene transfer in a rat fracture model

For the enhancement of fracture healing, either purified proteins or vectors for expression of growth factors in situ may be used. Adenoviral vectors directly convert cells to express a transgene. However, the cell types which are preferentially infected and the time of expression during fracture healing are currently not known. The adenoviral type 5 vectors used in this study are replication incompetent viruses, one encoding beta-galactosidase (beta-GAL) and one green fluorescent protein. Femora of 35 Sprague-Dawley rats were fractured. Three days after stabilization with Kirschner wire, 10(12) pfu viral suspension were injected into the fracture zone. As a control, five animals received injections of adenovirus type 2. Animals were sacrificed after 3 days, 1, 2 and 4 weeks. Fractures healed radiographically within 2-3 weeks. All specimens were examined for beta-GAL and green fluorescent protein (GFP) expression. Fibroblast and osteoblasts within callus tissue displayed a high transgene expression (week 1). A decrease of expression was observed during the observation period. In this experimental study, we have demonstrated that all cells of the primary callus can be transfected using adenoviral vectors, which provide a tool to further investigate adenoviral transfer of growth factors such as bone morphogenetic protein-2 (BMP-2).     

Adenoviral gene therapy, radiation, and prostate cancer

Viral gene therapy has exceptional potential as a specifically tailored cancer treatment. However, enthusiasm for cancer gene therapy has varied over the years, partly owing to safety concerns after the death of a young volunteer in a clinical trial for a genetic disease. Since this singular tragedy, results from numerous clinical trials over the past 10 years have restored the excellent safety profile of adenoviral vectors. These vectors have been extensively studied in phase I and II trials as intraprostatically administered agents for patients with locally recurrent and high-risk local prostate cancer. Promising therapeutic responses have been reported in several studies with both oncolytic and suicide gene therapy strategies. The additional benefit of combining gene therapy with radiation therapy has also been realized; replicating adenoviruses inhibit DNA repair pathways, resulting in a synergistic sensitization to radiation. Other, nonreplicating suicide gene therapy strategies are also significantly enhanced with radiation. Combined radiation/gene therapy is currently being studied in phase I and II clinical trials and will likely be the first adenoviral gene therapy mechanism to become available to urologists in the clinic. Systemic gene therapy for metastatic disease is also a major goal of the field, and clinical trials are currently under way for hormone-resistant metastatic prostate cancer. Second- and third-generation "re-targeted" viral vectors, currently being developed in the laboratory, are likely to further improve these systemic trials.     

Adenoviral gene transfer to the neonatal rat pulmonary circulation

BACKGROUND: Gene delivery to the pulmonary circulation has been studied in adult animals, but has not been extensively investigated in neonates. METHODS: We tested the ability of recombinant, replication-defective adenovirus to transduce the pulmonary circulation when delivered by percutaneous ventricular puncture. Five-day-old rat pups were injected with 10(7) to 10(10) particles (approximately 10(5) to 10(8) pfu) in 30 micro l total volume. RESULTS: Using RT-PCR, we detected transgene expression in both lung and liver at all dosages. However, whereas only 1/6 pups injected with 10(7) particles had detectable expression, 8/9 pups in the two highest dose groups had detectable expression. In the highest dose group expression was approximately 5-fold greater in lung than liver, though in the lower dose groups no difference between lung and liver was found. Expression decreased by only 25% from day 4 through the last time point at day 28 in lung, whereas liver expression was undetectable in 7 of 9 samples on day 28. Histopathological examination demonstrated expression both within the media of large arteries and in small, peripheral arteries and capillaries, with a concentration of expression in the most distal areas of both the lungs and liver. No evidence of inflammation was seen. CONCLUSIONS: We conclude that the neonatal pulmonary circulation can be effectively transduced using systemic adenoviral vector injection, has more sustained expression than liver, and may be a target for therapeutic gene delivery.     

人HIF—1α的腺病毒表达载体的构建与分析

低氧诱导因子 1(hypoxiainduciblefactor 1,HIF 1)是由HIF 1α和HIF 1β组成的异源二聚体转录因子 ,在细胞的氧平衡过程中起重要作用。在应答低氧信号时 ,HIF 1α亚基表达水平上调 ,并通过激活参与细胞能量代谢、红血细胞生成以及血管生成的靶基因表达 ,达到保护局部缺 贫血细胞免于凋亡或死亡 ,而后者则是临床上影响大脑和脊椎神经损伤恢复的主要原因。为了达到基因治疗急性神经损伤的目的 ,我们构建了表达HIF 1α的重组腺病毒载体。实验表明 ,重组腺病毒可以在大肠杆菌中组装 ,并在HEK2 93T细胞中包装。包装后的HIF 1α重组腺病毒载体的病毒感染效率为 2× 10 1 3CFU ,外源基因HIF 1α在He1a细胞中的表达 6h后达到峰值。目前正在开展建立在此基础上的急性神经损伤动物模型试验。     

腺病毒载体在肿瘤靶向性基因治疗中的应用

近年来,腺病毒载体广泛应用于恶性肿瘤的靶向性基因治疗。对传统腺病毒载体的改造主要有以下策略：(1)通过改变腺病毒的嗜性(tropism),使之具有感染宿主细胞的靶向性;(2)在转录水平控制外源性基因的表达;(3)可选择性裂解肿瘤细胞的有复制能力的腺病毒(replication-competent adenovirus,RCA)。更多安全、高效的靶向性腺病毒载体将应用于肿瘤基因治疗中。