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PubMed2004

Overview of adeno-associated viral vectors.

The use of adeno-associated virus (AAV) as a gene transfer vector has been steadily increasing over the past several years. AAV vectors have been particularly useful for applications where sustained gene expression is required. Prolonged in vivo expression following AAV treatment has been seen in the liver (1,2), brain (3,4), skeletal muscle (5,6), lung (7,8), and hematopoietic stem cells (9,10) of animal models. Therapeutic benefit from AAV treatment has been shown in a number of preclinical models of disease, including animal models of coagulopathies (11,12), lysosomal storage diseases (13,14), vision defects (15,16), and amino acid disorders. Clinical trials using AAV for the treatment of hemophilia B have begun, and early reports from these trials have been promising. In this introductory chapter to AAV, we will provide a brief overview of the molecular biology of this virus, an overview of methods of vector production, and a brief summary of the use of alternate AAV serotypes. The following chapters will then focus on specific methods and techniques for AAV transduction of the organs listed previously.

Dependovirus↗
PubMed2002

Tropism-modified adenoviral and adeno-associated viral vectors for gene therapy.

One of the most rapidly advancing areas of gene therapy is vector development. For the majority of gene therapy procedures, efficient and selective transduction would provide safe and more effective treatments at optimal vector doses. Advances in vector targeting strategies have been rapid within the field of DNA-based viruses, particularly adenovirus (Ad) and more recently adeno-associated virus (AAV) based vectors. Vector targeting at the level of virus: cell interaction can be achieved using both non-genetic and genetic methodology. Non-genetic approaches typically utilise bispecific antibodies that both neutralise wild-type virus tropism and provide a new cell binding capacity. For genetic targeting strategies, the virus capsid can be engineered to express foreign ligands that target selected receptors in the absence or presence of additional modification to ablate the virus' natural tropism. This review covers technological advances that have led to targeting of Ad and AAV and highlights the potential for these 'designer' viruses for future gene-based therapeutics.

Adenoviridae↗
PubMed2005

Adeno-associated viral vectors for retinal gene transfer and treatment of retinal diseases.

Retinal gene transfer holds big promises for the treatment of inherited and non-inherited blinding diseases, such as retinitis pigmentosa or age-related macular degeneration. Key to the development of successful gene-based therapies for the eye are efficient tools for retinal gene transfer. Vectors based on adeno-associated viruses (AAV) are able to transduce robustly and persistently different retinal cell types of animal models after a single intraocular administration. Recombinant AAV (rAAV) vectors are versatile gene transfer tools in that capsid proteins from dozens of AAV serotypes can be easily interchanged, resulting in the creation of recombinant vectors with unique transduction properties. This has allowed successful proof-of-principle studies using rAAV-mediated gene transfer to restore retinal morphology and function in small and large animal models of retinal diseases. In addition, gene delivery using rAAV vectors in the eye seems to have appropriate biosafety characteristics to rapidly move it from bench to bedside. All the above aspects will be reviewed and discussed in detail below.

Angiogenesis Inhibitors↗
PubMed2006

Recombinant adeno-associated viral vector encoding human VEGF165 induces neomicrovessel formation in the adult mouse brain.

Delivery of therapeutic genes represents a fascinating possibility to accelerate injury-repairing process in tissues that are otherwise difficult to treat, such as cerebral ischemia. Current studies indicate that gene transfer-induced focal angiogenesis in the brain may provide an important therapeutic strategy. In the present study, we reported the efficacy of induction of angiogenesis with an adeno-associated virus (AAV) vector expressing the 165 amino acid isoform of vascular endothelial growth factor (VEGF165). We found AAV serotype 1 has more efficiency in transduction of the brain tissue than AAV serotype 2. Quantitative vessel counting showed that microvessels in AAV-VEGF transduced mice significantly increased from 1 week up to 12 weeks compared to the control groups (AAV-VEGF: 316+/-58 vs. AAV-lacZ: 180+/-34 and saline: 152+/-35 vessels/mm2, at 6 weeks, p<0.05). Proliferating cell nuclear antigen (PCNA) staining confirmed these microvessels were actively proliferating. Double-labeled fluorescence staining demonstrated that neurons, astrocytes, and endothelial cells could express VEGF following AAV-VEGF gene transfer. AAV vectors did not elicit a detectable inflammatory response, cell loss or neuronal damage. Our data underline the importance of angiogenesis in the brain tissue and indicate that VEGF gene transfer might present a valuable approach to treat brain ischemic disorders.

Animals↗
PubMed2004

[Gene transfer by novel non-viral vector polyethylenimine].

OBJECTIVE: To investigate the influencing factors of polyethylenimine (PEI) in gene transfer in vitro. METHODS: Cytotoxic effects of PEI on in vitro cultured NIH 3T3 cells were quantified by MTT assay. The interaction between PEI and DNA at different charge ratios was analyzed by agarose gel electrophoresis retardation assay. The expression of gene transfer was monitored in Cos-7 cells using pEGFP and pSV beta plasmids as the reporter gene systems. Influences of chloroquine, albumin, serum, salt ion strength, and Mg(2+) ion and other factors on PEI/DNA transfer efficiency were evaluated. RESULT: The survival rate of NIH3T3 cells at 6 mg/L of PEI was 64.2% and at 7 mg/L of PEI was 54.4%. Gel electrophoresis retardation assays showed that PEI completely retarded DNA migration at 3.0 PEI nitrogen per DNA phosphate. Chloroquine enhanced the transfection efficiency of PEI. Albumin and serum in the culture medium decreased the transfection efficiency. HBS(HEPES buffered solution) or 150 mmol/L NaCl as the dilution solution of PEI/DNA was superior over 278 mmol/L glucose solution in the transfection efficiency. Mg(2+) in the dilution solution decreased the transfer efficiency of PEI/DNA. CONCLUSION: PEI is efficient gene transfer agent of eukaryotes in vitro, and can be possibly used in vivo.

Animals↗
PubMed2000

Adeno-associated viral vectors as gene delivery vehicles.

Adeno-associated virus (AAV), a non-pathogenic human parvovirus, is gaining attention for its potential use as a human gene therapy vector. One of the most attractive features of recombinant AAV vectors is the ability to be stably maintained in host cells as integrated proviruses. This property is particularly desireable for therapies requiring long-term correction of a genetic defect. This review highlights recent advances made in the AAV field and will discuss some limitations of rAAV vector integration. A novel method for enhancing the integration efficiency of these vectors will be presented.

Animals↗
PubMed2003

Cutting edge: a potent adjuvant effect of ligand to receptor activator of NF-kappa B gene for inducing antigen-specific CD8+ T cell response by DNA and viral vector vaccination.

The ligand to receptor activator of NF-kappaB (RANK-L)/RANK interaction has been implicated in CD40 ligand/CD40-independent T cell priming by dendritic cells. In this report, we show that the coadministration of the RANK-L gene with a Trypanosoma cruzi gene markedly enhances the induction of Trypanosoma Ag-specific CD8(+) T cells and improves the DNA vaccine efficacy. A similarly potent adjuvant effect of the RANK-L gene on the induction of Ag-specific CD8(+) T cells was also observed when recombinant influenza virus expressing murine malaria Ag was used as an immunogen. In contrast, the coadministration of the CD40L gene was not effective in these systems. Our results demonstrated, for the first time, the potent immunostimulatory effect of the RANK-L gene to improve the CD8(+) T cell-mediated immunity against infectious agents.

Adjuvants, Immunologic↗
PubMed2006

Suppressive effect of modified dominant negative RAS mutant on human cancer by gene transfer with non-viral vector.

N116Y, H-RAS mutant, has dominant negative activity in the RAS function and a suppressive effect on the growth of various types of cancer cells. However, a replication error of N116Y is of potential concern for carcinogenesis in clinical application. To decease the concern, we constructed modified N116Y by deleting the carboxyl terminus, which is necessary for the oncogenic function of Ras. One of the C-terminal deletion mutants of N116Y, N116Y-Cdel2 showed a growth-suppressing effect on various human cancer cell lines in vitro: the cervical cancer cell line HeLa, the pancreatic cancer cell line PCI43, the colon cancer cell lines SW480 and LoVo, and the tongue cancer cell line SAS. In addition, the suppressive effect of N116Y-Cdel2 on LoVo cells was also observed in vivo using a nonviral gene transfer vector, HVJ envelope. Our experiments suggest that the modified N116Y is a potential candidate gene for human cancer gene therapy.

Gene Transfer Techniques↗
PubMed1999

Development of viral vectors for gene therapy of beta-chain hemoglobinopathies: optimization of a gamma-globin gene expression cassette.

Progress toward gene therapy of beta-chain hemoglobinopathies has been limited in part by poor expression of globin genes in virus vectors. To derive an optimal expression cassette, we systematically analyzed the sequence requirements and relative strengths of the Agamma- and beta-globin promoters, the activities of various erythroid-specific enhancers, and the importance of flanking and intronic sequences. Expression was analyzed by RNase protection after stable plasmid transfection of the murine erythroleukemia cell line, MEL585. Promoter truncation studies showed that the Agamma-globin promoter could be deleted to -159 without affecting expression, while deleting the beta-globin promoter to -127 actually increased expression compared with longer fragments. Expression from the optimal beta-globin gene promoter was consistently higher than that from the optimal Agamma-globin promoter, regardless of the enhancer used. Enhancers tested included a 2.5-kb composite of the beta-globin locus control region (termed a muLCR), a combination of the HS2 and HS3 core elements of the LCR, and the HS-40 core element of the alpha-globin locus. All three enhancers increased expression from the beta-globin gene to roughly the same extent, while the HS-40 element was notably less effective with the Agamma-globin gene. However, the HS-40 element was able to efficiently enhance expression of a Agamma-globin gene linked to the beta-globin promoter. Inclusion of extended 3' sequences from either the beta-globin or the Agamma-globin genes had no significant effect on expression. A 714-bp internal deletion of Agamma-globin intron 2 unexpectedly increased expression more than twofold. With the combination of a -127 beta-globin promoter, an Agamma-globin gene with the internal deletion of intron 2, and a single copy of the HS-40 enhancer, gamma-globin expression averaged 166% of murine alpha-globin mRNA per copy in six pools and 105% in nine clones. When placed in a retrovirus vector, this cassette was also expressed at high levels in MEL585 cells (averaging 75% of murine alpha-globin mRNA per copy) without reducing virus titers. However, recombined provirus or aberrant splicing was observed in 5 of 12 clones, indicating a significant degree of genetic instability. Taken together, these data demonstrate the development of an optimal expression cassette for gamma-globin capable of efficient expression in a retrovirus vector and form the basis for further refinement of vectors containing this cassette.

Anemia, Sickle Cell↗
PubMed1999

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.

Animals↗
PubMed2001

Intraneoplastic polymer-based delivery of cyclophosphamide for intratumoral bioconversion by a replicating oncolytic viral vector.

rRp450 is an oncolytic herpesvirus that expresses the CYP2B1 cDNA, responsible for bioconverting cyclophosphamide (CPA) into the active metabolites 4-hydroxyCPA/aldophosphamide (AP). However, formal proof of prodrug activation is lacking. We report that activation of CPA in cells infected with rRp450 generates a time-dependent increase of diffusible 4-hydroxyCPA/AP. For in vivo applications, a CPA-impregnated polymer was implanted into human tumor xenografts inoculated with rRp450. The area under the curve for 4-hydroxyCPA/AP was 806 microg/g of tumor tissue/h when CPA was administered via intraneoplastic polymer and 3 microg/g of tumor tissue/h when CPA was administered i.p. Therefore, (a) a lytic virus expressing a "suicide" gene can activate a prodrug; and (b) within rRp450-infected tumor, more prolonged and higher concentrations of activated metabolites are generated by intraneoplastic compared with systemic administration of prodrug.

Animals↗
PubMed2002

RNAi and viral vectors as useful tools in the functional genomics of plants. Construction of BMV-based vectors for RNA delivery into plant cells.

The sequencing of several complete genomes and the development of a DNA microarray technology are among the most important achievements of molecular biology. They gave the proper grounds for the development of modern functional genomics. However, there is one additional condition which needs to be satisfied to truely enable the study of how a genome works: a suitable method of selectively inducing and silencing the expression of each individual gene. The methods used so far have usually only permitted the influencing of gene expression through genetic manipulations at the DNA level (genetically modified plants). The discovery of RNA interference (RNAi) opens up completely new possibilities of research on the functioning of particular plant genes, without the necessity of altering the genome structure. In this case, interference takes place at the transcript level. Thus, at any given moment during plant development, the expression of a specific gene (or several genes) can be inhibited, even if it is important for the survival of the organism under study. To this end, a double-stranded RNA inducing the RNAi phenomenon has to be delivered into the plant cell. Here we describe the construction of four brome mosaic virus-based vectors, which, as our preliminary data indicate, can be used to transfer RNA into barley cells.

Bromovirus↗
PubMed2003

Biophysical characterization of an integrin-targeted non-viral vector.

BACKGROUND: The formulation of polycationic complexes containing plasmid DNA for optimal transfection in vitro and in vivo for DNA vaccination, gene therapy and other applications continues to be a major research goal. Here we present new data on the biophysical properties of an integrin-targeted plasmid DNA (LID) formulation. MATERIAL/METHODS: Two plasmids (D), pEGFP (4.7 kb) and pCI-luc (5.7 kb), were mixed with a synthetic a5b1 integrin-targeted peptide (I), [K]16 GACRRETAWACG, in the presence of a cationic liposome (L), Lipofectin, composed of DOTMA and DOPE to form LID complexes. The physical properties of the complexes were measured using a variety of techniques including dynamic light scattering and fluorescence methods. The in vitro gene delivery to neuroblastoma cells with LID complexes was also assessed. RESULTS: We demonstrate the effects of complex size and charge ratio on in vitro transfection of mouse (Neuro-2A) and human (IMR-32) neuroblastoma cells. We report a significant increase in the level of luciferase and green fluorescent protein expression when transfection is performed in buffers of physiological ionic strength and hypothesise that the enhancement in transfection is caused by an increase in the size of the complexes observed during mixing and maturation. CONCLUSIONS: Cell transfection is also shown to be dependent on complex size and charge ratio, with large complexes prepared at charge ratios above 4.0 demonstrating efficient transfection.

Animals↗