Tropism-modification strategies for targeted gene delivery using adenoviral vectors

Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans) and "bridging" interactions. "Bridging" interactions refer to coagulation factor binding, namely coagulation factor X (FX), which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs). These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad) have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon), pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR) substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies), can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of "bridging interactions" such as those which occur between the hexon of Ad5 and coagulation factor X (FX), or alternatively, through the use of polymer-coated "stealth" vectors which avoid these interactions. Simultaneous retargeting and detargeting can be achieved by combining multiple genetic and/or chemical modifications.     

In vivo marking of rhesus monkey lymphocytes by adeno-associated viral vectors: direct comparison with retroviral vectors.

We have compared adeno-associated virus (AAV)-based and retrovirus-based vectors for their ability to transduce primary T lymphocytes in vitro and then tracked the persistence of these genetically marked lymphocytes in vivo, using the rhesus monkey model. To avoid the complication of immune rejection of lymphocytes transduced with xenogeneic genes in tracking studies primarily designed to investigate transduction efficiency and in vivo kinetics, the vectors were designed without expressed genes. All vectors contained identically mutated beta-galactosidase gene (beta-gal) and neomycin resistance gene (neo) DNA sequences separated by different length polylinkers, allowing simple differentiation by polymerase chain reaction (PCR). Each of 2 aliquots of peripheral blood lymphocytes from 4 rhesus monkeys were transduced with either AAV or retroviral vectors. The in vitro transduction efficiency (mean vector copy number/cell) after the ex vivo culture was estimated by PCR at 0.015 to 3.0 for AAV, varying depending on the multiplicity of infection (MOI) used for transduction, and 0.13 to 0.19 for the retroviral transductions. Seven days after transduction, Southern blot analysis of AAV-transduced lymphocytes showed double-stranded and head-to-tail concatemer forms but failed to show integration of the AAV vector. AAV and retroviral aliquots were reinfused concurrently into each animal. Although the retrovirally marked lymphocytes could be detected for much longer after infusion, AAV transduction resulted in higher short-term in vivo marking efficiency compared with retroviral vectors, suggesting possible clinical applications of AAV vectors in lymphocyte gene therapy when long-term vector persistence is not required or desired.     

Direct gene delivery to synovium: An evaluation of potential vectors in vitro and in vivo

Objective. To assess the abilities of various vectors to transfer genes to the synovial lining of joints. Methods. Vectors derived from retrovirus, adenovirus, and herpes simplex virus as well as cationic liposomes and naked plasmid DNA were evaluated. Each construct contained the lac Z marker gene; and one retroviral construct, and one plasmid also contained a gene encoding human interleukin-1 receptor antagonist. Gene expression was under the control of the human cytomegalovirus promoter in all vectors except the retrovirus, where the endogenous 5′ long terminal repeat was retained as the promoter. Cultures of rabbit synovial fibroblasts were exposed to these vectors and stained with X-gal to identify lac Z+ cells. Vectors were then injected directly into rabbits' knee joints, and gene transfer and expression were assessed by X-gal staining and polymerase chain reaction (PCR). Results. Adenovirus was a highly effective vector both in vitro and in vivo, with lac Z gene expression persisting for at least 28 days. However, an inflammatory response was noted in vivo. Cells infected in vitro and in vivo with herpes simplex virus also expressed the lac Z gene at high levels, but expression was limited by cytotoxicity. Retroviruses, in contrast, were effective only under in vitro conditions, permitting cell division. Liposomes gave variable in vitro results; when injected into joints in vivo, gene expression was low and was detectable for only a few days, even though a PCR signal persisted for at least 28 days. Unexpectedly, plasmid DNA was captured by the synoviocytes and expressed transiently following intraarticular injection. Conclusion. None of the vectors was ideal for in vivo gene delivery to synovium, although adenovirus was clearly the most effective of those tested. Retroviruses, although poor vectors for in vivo gene delivery, are well suited for ex vivo gene transfer to the synovial lining of joints.     

In vitro and in vivo anti-tumor activities of anti-EGFR single-chain variable fragment fused with recombinant gelonin toxin

Purpose

Epidermal growth factor receptor (EGFR) plays an important role in the growth and metastasis of many solid tumors. Strategies that target EGFR hold promising therapeutic potential for the treatment for non-small cell lung cancer (NSCLC), as EGFR is normally overexpressed in these tumors. This study was designed to determine whether an anti-EGFR immunotoxin has anti-tumor activity against NSCLC, and if so, to further investigate the possible mechanisms of cytotoxicity.

Methods

A fusion protein of anti-EGFR single-chain variable fragment (anti-EGFR scFv) and the plant toxin gelonin (rGel) was constructed, expressed in bacteria, and purified to homogeneity. Cytotoxicity of anti-EGFR scFv/rGel (E/rG) immunotoxin was assessed on A549, HCC827, and H1975 cells (EGFR-overexpressing NSCLC-derived cell lines) and A549 xenografts in nude mice.

Results

Cytotoxicity experiments using E/rG on A549, HCC827, and H1975 cells demonstrated that E/rG can specifically inhibit proliferation of these cells, whereas it did not affect the proliferation of Raji cells that do not express EGFR. Treatment for A549 xenografts in nude mice with E/rG resulted in significant suppression of tumor growth compared to controls. Immunofluorescence in frozen tissue sections confirmed that E/rG could specifically bind to tumor tissues in nude mice bearing A549 tumor xenografts, while rGel alone showed no binding activity. Furthermore, E/rG inhibited the growth of A549 cells by cytotoxic effects that blocked tumor proliferation, and the immunotoxin-induced cell death may be mediated by autophagy.

Conclusions

These results showed that E/rG might have significant potential as a novel clinical therapeutic agent against human NSCLC.     

Liver-directed gene therapy: quantitative evaluation of promoter elements by using in vivo retroviral transduction.

Liver-directed gene therapy will be applicable tomany inherited diseases. Although various protocols have been devised for invivo delivery of retrovirus, comparison of hepatocyte transduction frequencieshas been difficult due to variations in retroviral titer and a paucity of DNAdata. We have previously reported an in vivo rat hepatocyte transductiontechnique which involves 70% hepatectomy followed 24 hr later by portal veininjection of retrovirus during hepatic in-flow occlusion. In this study, weemployed this method and concentrated retroviral preparations to achievetransduction of up to 15% of hepatocytes as determined by a quantitative PCRassay. As an initial step toward identifying promoters which lead to high-levellong-term expression of retroviral transduced genes, we used our in vivodelivery system to compare the Moloney murine leukemia virus long terminalrepeat (LTR) promoter with the promoter for the large subunit of murine RNApolymerase II (Pol-II). Human alpha 1-antitrypsin (hAAT) was used as thereporter gene to facilitate long-term analysis of expression. Serum hAAT levelswere higher for the Pol-II promoter (143 ng/ml) than for the LTR promoter (50ng/ml). This difference was consistent with the higher transduction frequencyobserved for the Pol-II-hAAT vector. Although serum hAAT expression wassustained for up to 1 year in six of eight Pol-II-hAAT-transduced rats and threeof five LTR-hAAT-transduced rats and was proportional to hAAT mRNA level andproviral DNA frequency, in vivo expression was significantly lower than intransduced tissue culture cells. We conclude that a high frequency of in vivotransduction can be achieved by using retroviral vectors and our rapidtransduction protocol, but transduced gene expression remains a serious problem.The quantitative assays described herein will facilitate in vivo comparisons ofgene regulatory elements.     

非复制型腺病毒介导抗-HBc单链抗体的细胞内表达

目的探讨非复制型腺病毒介导抗-HBc单链抗体(ScFv)细胞内表达效率,并确定是否具有特异性抗原结合活性。方法将经细菌内同源重组并在293细胞中包装产生的携带抗HBc ScFv基因的非复制型重组腺病毒,按感染复数为10体外转染HepG2细胞,荧光显微镜下观察绿色荧光蛋白的表达；取培养上清液和细胞裂解上清液进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳和Western blot法检测HBc ScFv表达。结果重组腺病毒Ad-ScFv感染HepG2细胞后,荧光显微镜下观察到HepG2细胞中绿色荧光蛋白；十二烷基硫酸钠聚丙烯酰胺凝胶电泳显示培养上清液和细胞裂解物中可见2.7×10~4左右的蛋白条带；Western blot显示有乙型肝炎核心抗原特异性结合活性阳性反应条带出现。结论携带抗-HBc ScFv基因的非复制型重组腺病毒能在真核细胞中有效表达抗-HBc ScFv,并具有特异性抗原结合活性。     

Epstein-Barr virus vectors for gene delivery to B lymphocytes.

Basic research in Epstein-Barr virus (EBV) molecular genetics has provided means to maintain episomes in human cells, to efficiently deliver episomes with up to 150 kbp of heterologous DNA to human B lymphocytes, and to immortalize human B lymphocytes with EBV recombinants that can maintain up to 120 kbp of heterologous DNA. Episome maintenance requires an EBV nuclear protein, EBNA1, whereas immortalization of cells with EBV recombinants requires EBNA1, EBNA2, EBNA3A, EBNA3C, EBNALP, and LMP1. EBV-derived vectors are useful for experimental genetic reconstitution in B lymphocytes, a cell type frequently used in establishing repositories of human genetic deficiencies. The ability of EBV-infected cells to establish a balanced state of persistence in normal humans raises the possibility that cells infected with EBV recombinants could be useful for genetic reconstitution, in vivo.     

Aerosol gene delivery in vivo.

The ability to express transgenes selectively within the lung will greatly facilitate the development of gene therapy for a variety of human diseases. We have demonstrated that aerosol administration of a chloramphenicol acetyltransferase (CAT) expression plasmid complexed to cationic liposomes produces high-level, lung-specific CAT gene expression in mice in vivo. Significant levels of CAT activity are seen in the lungs for at least 21 days following aerosolization. In situ immunostaining for intracellular CAT protein reveals that the majority of airway epithelial and alveolar lining cells are transfected in vivo. Histological analyses show no apparent treatment-related damage. These results have important implications for the development of human gene therapy.     

Ex vivo gene delivery using an adenovirus vector in treatment for cartilage defects

OBJECTIVE: To realize local selective gene expression in grafted chondrocytes for cartilage defect, we investigated the usefulness of an ex vivo gene delivery method using an adenovirus vector. METHODS: Beta-galactosidase gene (LacZ) was transfected using an adenovirus vector to chondrocytes isolated from rat joints. The cells were then embedded into collagen gel, and LacZ expression in the gel was examined using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining; beta-galactosidase activity was also measured. The collagen gel containing transfected chondrocytes was grafted to the experimental cartilage defects, and the expression of delivered gene was histologically examined after X-gal staining of the tissue containing the grafted area. RESULTS: X-gal positive chondrocytes in the gel accounted for 82% at one week and 55% at 8 weeks after gene delivery. Beta-galactosidase activity decreased with time, but its expression was maintained even at 8 weeks after gene delivery. Chondrocytes used in the allograft maintained their morphology, and the expression of delivered gene continued during the 8 week period. CONCLUSION: In this ex vivo method, delivered gene can be expressed efficiently for a long time; this method would be useful in allografts for cartilage defects.     

Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes

Achieving efficient in vivo delivery of siRNA to the appropriate target cell would be a major advance in the use of RNAi in gene function studies and as a therapeutic modality. Hepatocytes, the key parenchymal cells of the liver, are a particularly attractive target cell type for siRNA delivery given their central role in several infectious and metabolic disorders. We have developed a vehicle for the delivery of siRNA to hepatocytes both in vitro and in vivo, which we have named siRNA Dynamic PolyConjugates. Key features of the Dynamic PolyConjugate technology include a membrane-active polymer, the ability to reversibly mask the activity of this polymer until it reaches the acidic environment of endosomes, and the ability to target this modified polymer and its siRNA cargo specifically to hepatocytes in vivo after simple, low-pressure i.v. injection. Using this delivery technology, we demonstrate effective knockdown of two endogenous genes in mouse liver: apolipoprotein B (apoB) and peroxisome proliferator-activated receptor alpha (ppara). Knockdown of apoB resulted in clear phenotypic changes that included a significant reduction in serum cholesterol and increased fat accumulation in the liver, consistent with the known functions of apoB. Knockdown of ppara also resulted in a phenotype consistent with its known function, although with less penetrance than observed in apoB knockdown mice. Analyses of serum liver enzyme and cytokine levels in treated mice indicated that the siRNA Dynamic PolyConjugate was nontoxic and well tolerated.     

Role of melanotransferrin in iron metabolism: studies using targeted gene disruption in vivo

Melanotransferrin (MTf) or tumor antigen p97 is a transferrin homolog that binds one iron (Fe) atom and has been suggested to play roles in a variety of processes, including Fe metabolism, eosinophil differentiation, and plasminogen activation. Considering the vital role of Fe in many metabolic pathways, such as DNA and heme synthesis, it is important to understand the function of MTf. To define this, a MTf knockout (MTf-/-) mouse was generated through targeted disruption of the MTf gene. The MTf-/- mice were viable and fertile and developed normally, with no morphologic or histologic abnormalities. Assessment of Fe indices, tissue Fe levels, hematology, and serum chemistry parameters demonstrated no differences between MTf-/- and wild-type (MTf+/+) mice, suggesting MTf was not essential for Fe metabolism.     

The use of lentiviral vectors in gene therapy of leukemia: combinatorial gene delivery of immunomodulators into leukemia cells by state-of-the-art vectors

Our goal is to develop cell vaccines against leukemia cells, genetically modified to express molecules with potent immune-stimulatory capacities. Pre-clinical evaluation of this approach in murine models has demonstrated efficient anti-leukemic responses with the expression of immunomodulators, in particular GM-CSF and CD80, in irradiated cell vaccines. We have previously shown efficient insertion of GM-CSF and CD80 genes into primary human leukemia cells with the use of second and third generation self-inactivating (SIN) lentiviral vectors (Blood 96 (2000), 1317; Leukemia 16 (2002), 1645). The advantages of lentiviral vectors for development of autologous leukemia cell vaccines include: (1) efficient and consistent gene delivery; (2) high levels of transgene expression; (3) persistent expression of the transduced gene; (4) no viral proteins, as only the transduced gene is expressed; (5) no undesirable cytotoxic effects, and; (6) simplicity of use [leukemia cells are exposed to vector(s) only once]. In this work, we evaluated the insertion of the central polypurine tract and the central termination sequence into a SIN lentiviral vector encoding for GM-CSF and CD80, which significantly enhanced the transduction efficiency of primary leukemia cells and provided higher levels of GM-CSF and CD80 co-expression. We also demonstrate a methodology to deliver simultaneously a combination of immunomodulatory molecules (GM-CSF, CD80, IL-4, and CD40L) to activate different pathways of immune stimulation. Therefore, lentiviral vectors offer a simple, versatile, and reliable approach for engineering leukemic cells for use as cell vaccines.     

Effects of CD2 locus control region sequences on gene expression by retroviral and lentiviral vectors.

Locus control region (LCR) sequences are involved in the establishment of open chromosomal domains. To evaluate the possibility of exploiting the human CD2 LCR to regulate gene expression by Moloney murine leukemia virus (Mo-MLV)-based retroviral vectors in T cells, it was included in vectors carrying the enhanced green fluorescence protein (EGFP) reporter gene; then transduction in vitro of lymphoid and nonlymphoid cell lines was performed. Deletion of the viral enhancer in the Mo-MLV long terminal repeat was necessary to detect LCR activity in the context of these retroviral vectors. It was found that a full-length (2.1 kb), but not a truncated (1.0 kb), CD2 LCR retained the ability to modulate reporter gene expression by Mo-MLV-derived retroviral vectors, leading to a homogeneous, unimodal pattern of EGFP expression that remained unmodified in culture over time, specifically in T-cell lines; on the other hand, viral titer was strongly reduced compared with vectors not carrying the LCR. Lentiviral vectors containing the CD2 LCR could be generated at higher titers and were used to analyze its effects on gene expression in primary T cells. Subcutaneous implantation of genetically modified cells in immunodeficient mice showed that retroviral vectors carrying the CD2 LCR conferred an advantage in terms of transgene expression in vivo, compared with the parental vector, by preventing the down-modulation of EGFP expression. These findings suggest a potential application of this LCR to increase gene expression by retroviral and lentiviral vectors in T lymphocytes.     

Generation of packaging cell lines for pseudotyped retroviral vectors of the G protein of vesicular stomatitis virus by using a modified tetracycline inducible system.

We have previously shown that the G protein of vesicular stomatitis virus (VSV-G) can be incorporated into the virions of retroviruses. Since expression of VSV-G is toxic to most mammalian cells, development of stable VSV-G packaging cell lines requires inducible VSV-G expression. We have modified the tetracycline-inducible system by fusing the ligand binding domain of the estrogen receptor to the carboxy terminus of a tetracycline-regulated transactivator. Using this system, we show that VSV-G expression is tetracycline-dependent and can be modulated by beta-estradiol. Stable packaging cell lines can readily be established and high-titer pseudotyped retroviral vectors can be generated upon induction of VSV-G expression.     

A system for tissue-specific gene targeting: transgenic mice susceptible to subgroup A avian leukosis virus-based retroviral vectors.

Avian leukosis viruses (ALVs) have been used extensively as genetic vectors in avian systems, but their utility in mammals or mammalian cell lines is compromised by inefficient viral entry. We have overcome this limitation by generating transgenic mice that express the receptor for the subgroup A ALV under the control of the chicken alpha sk-actin promoter. The skeletal muscles of these transgenic animals are susceptible to efficient infection by subgroup A ALV. Because infection is restricted to cell lineages that express the transgene, the method has utility for studies of development and oncogenesis and will provide models for tissue-specific gene therapy.