The vector systems currently available through Penn Vector Core are those derived from adeno-associated virus (AAV), adenovirus and lentivirus. The Core offers AAV-based vectors pseudotyped with a variety of different serotype capsids which have demonstrated transduction profiles superior to those of previous generations in a variety of target tissues. The Core currently provides AAV vectors based on serotypes 1, 2, 5, 6, 7, 8, 9, and rh10 with additional serotypes under evaluation. Adenoviral vectors generated by the Core include those with a variety of backbone deletions including E1-deleted, E3-deleted and E1- and E3-deleted vectors. Lentiviral vectors pseudotyped with a range of envelope glycoproteins such as those derived from VSV-G, MuLV, LCMV, Mokola and deletion variants of Ebola are also available to investigators through Penn Vector Core. For details on each vector system provided, please follow the links below:
Adeno-Associated Viral Vectors (AAV) Recombinant adeno-associated virus, a nonpathogenic human parvovirus, is one of the most promising viral vectors for gene transfer. AAV is a single-stranded DNA-containing virus, which has not been shown to be associated with any known symptoms or pathology in humans to date. The wide host range of the virus, the long-term expression in vivo, and the low immunogenicity have contributed significantly toward the development of AAV vectors as a useful alternative to the more commonly used retroviral and adenoviral vectors.
Small, routine and large scale preparations of AAV vectors are generated following classic triple transfection of subconfluent HEK293 cells by three plasmids: AAV cis-plasmid encoding the gene of interest, AAV trans-plasmid containing AAV rep and cap genes, and adenovirus helper plasmid, pDF6. The culture medium containing the vector particles is collected, concentrated using tangential flow filtration, purified by iodixanol gradient ultracentrifugation, further concentration and buffer exchange. Purified vector preparations are subjected to Quality Control appropriate for preclinical studies in large or small animal models.
AAV2: AAV2 is the most widely studied AAV serotype, and many in vitro and in vivo studies have been performed based on this serotype (Kotin, 1994). Despite the impressive longevity of transgene expression obtained with AAV2, its application has been limited because of low level of transgene expression. Blocks at the level of vector entry and post-entry processing contribute to these inefficiencies (Sanlioglu et al., 2001). In addition, the prevalence of preexisting antibodies against AAV2 in human populations (30% are serum positive in some populations) could hinder the application of AAV2 as a gene transfer vector in humans (Chirmule et al., 1999).
Novel AAV Serotypes: Progress in overcoming these barriers has been made through the development of vectors based on other serotypes that enter the cell via receptors distinct from those that recognize AAV2 and that are not affected by the presence of preexisting anti-AAV2 antibodies. Several other serotypes of AAV (i.e., serotypes 1, 3, 4, 5, and 6) had been isolated and vectors based on these alternative serotypes developed (Kay et al., 2001). More recently, additional novel AAV serotypes were isolated from the tissues of human and non-human primates in the laboratory of Dr. James Wilson (University of Pennsylvania) and developed as vectors The in vivo performance of vectors derived from these novel serotypes, particularly those based on AAV7, 8, 9 and rh10, has been found to far exceed that of AAV2 in certain target tissues (Gao et al., 2005 New Recombinant serotypes of AAV vectors)
AAV Serotype Vector Tropism: Penn Vector Core currently offers AAV vectors based the AAV serotypes 1, 2, 5, 6, 7, 8, 9, rh10 with additional serotype AAV vectors under development. The tropism of selected AAV serotype vectors is shown in the following table however it should be noted that performance may be dependent upon animal model, age and route of administration among other variables.
|AAV1||Muscle, heart, CNS|
|AAV2||In vitro, CNS|
|AAV5||Lung (airway, alveoli), eye, CNS|
|AAV6||Lung (airway), heart|
|AAV8||Liver, muscle, eye, CNS|
|AAV9||Lung (alveoli), liver, muscle, heart, CNS|
- Genome Titer Determination
- Endotoxin Assay
- Infectivity Assay (TCID50)
- Purity Assessment (SDS-PAGE)
Adenoviral Vectors: Recombinant adenoviral vectors are one of the most efficient viral vectors for gene delivery, both in vivo and in vitro, due to their high transduction efficiency, broad host range, ability to infect non-dividing cells, and potential for generating high titer virus. Applications for this vector include gene therapy, particularly in cases where high level, short term gene expression is required, vaccine applications and basic gene transfer studies. Recombinant adenoviral vectors are produced by direct cloning, produced in complementing cell lines and purified by cesium chloride gradient ultracentrifugation.
Creation of Recombinant Adenoviral Vector
Description of Method: Flowchart of Direct Ligation (PDF download)
We have developed a direct cloning and green-white selection process for use at the level of bacterial transformants. Plasmid constructs containing a recombinant adenovirus genome that can be rescued are created by transfection into the appropriate cell line. The method is based on Clontech's Adeno-X Expression SystemTM, and has been further refined and modified by the Vector Development Laboratory. The green-white selection method for homologous recombination significantly simplifies the procedure for isolation of recombinant adenoviruses; however, the desired recombinants are usually contaminated with other adenovirus species, which requires a lengthy and tedious plaque purification and re-characterization process. In our recent direct cloning modification, the cDNA encoding the enhanced green fluoroescent protein (EGFP), driven by the bacterial LacZ promoter, and flanked by two introns coding rare restriction enzyme sites, is inserted into the E1-region of an adenoviral vector genome carrying a plasmid backbone with an ampicillin selective marker. The gene of interest is first cloned into a shuttle plasmid with a kanamycin marker between a mammalian promoter and polyadenylation signal. The transgene cassette in the shuttle plasmid flanked by the same rare restriction sites as in the adenovirus backbone plasmid is isolated by treatment with these enzymes and ligated into the viral backbone plasmid, which is digested with the same enzymes. As a result of the ligation reactions, the bacterial transformants will be double selected with ampicillin resistance and fluorescent microscopy. Over 90% of the white colonies contain the correct recombinant adenoviral vector genomes. Thus, a homogeneous recombinant adenoviral vector population is generated by treating the plasmid with PacI to release both ITRs of the adenoviral vector genome, followed by transfection and rescue in appropriate cell lines. Since there is no need for plaque purification, recombinant adenoviral vectors can be created with this highly efficient method in as little as three weeks. In addition, the flexibility in the introduction of deletions and mutations into the adenoviral vector backbone plasmid makes it easy to create a variety of recombinant viruses with different backbones.
- Particle Number Determination
- Endotoxin Assay
- Infectivity Assay (TCID50)
- DNA Structure Analysis
- Replication Competent Adenovirus (RCA) Assay
Lentiviral Vectors: Lentiviruses have the ability to transduce a wide variety of cells, including nondividing cells, and to integrate into the genome to provide sustained gene expression. Recently, it has been demonstrated that changing the pseudotype of lentiviruses can modify their tissue tropism. The surface glycoprotein from Rhabdovirus vesicular stomatitis virus (VSV-G) is commonly used for pseudotyping retroviruses because it is highly stable and confers an exceptionally wide host range, because of the binding of VSV-G to a cell surface lipid. However, not all tissues can be efficiently transduced by VSV-G pseudotyped lentiviruses, such as airway epithelia. A panel of viral envelopes was evaluated for their ability to pseudotype HIV-based vector and transduce airway epithelia cells, mouse brain, human bone marrow, fetal muscle and fetal liver in experiments performed by several Penn investigators. Their studies demonstrated that an envelope derived from the Zaire strain of the Ebola virus (EboZ) conferred strong ability to transduce human airway epithelium in vitro and ex vivo. Targeted transduction of specific brain regions can be achieved with pseudotyped lentiviral vectors. Recently, a collection of the glycoprotein from a variety of different enveloped viruses, including Ebola virus (Zaire and Reston subtypes), Marburg virus, rabies virus, LCMV, Mokola virus, influenza virus (HA), murine leukemia virus (amphotropic envelope), and respiratory syncytial virus (RSV) have been transferred to the Vector Core from Dr. Wilson's lab. These large-scale productions of pseudotyped lentiviral vectors were added as new Vector Core services.
Currently, the following six viral envelops are available through Vector Core: VSVG, MuLV, LCMV, EboZ-GP, NTDL6 (EboZ-GP with deletions), and Mokola.
Large-scale production of pseudotyped lentiviral vector by triple transfection and concentration by ultracentrifugation:
Pseudotyped lentiviral vector is produced by transfection of 20 x 150 mm plates of 293T cells with three plasmids: the helper packaging construct pCMVDR8.2, the transfer vector, and the plasmid encoding for envelope protein using the calcium phosphate precipitation method. At 44h after transfection, fresh medium is added to each plate for 16h before collection of virus. The medium containing viruslike particles is filtered through a 0.45mm filter and concentrated by ultracentrifugation for 2h at 4°C. Virus is resuspended in complete DMEM, aliquoted, and stored at -80°C.
QC assays for lentiviral vectors:
For GFP or LacZ containing vectors, infectious titer is determined by transduction on 293T cells and analyzed by fluorescence microscopy or by X-gal staining. p24 is determined by an ELISA assay. Some of the viral stocks are also tested for the presence of replication-competent lentivirus by monitoring p24 antigen expression in the culture medium of transduced 293T cells for 30 days. In addition, a quantitative RT-PCR based assay is currently under development for genome titration.
- Infectious Titer Determination
- Endotoxin Assay
- Replication Competent Lentivirus (RCL) Assay
- Mochizuki H, Schwartz JP, Tanaka K, Brady RO, Reiser J. 1998. High-titer human immunodeficiency virus type 1-based vector systems for gene delivery into nondividing cells. J Virol, 72(11):8873-83.
- Kobinger GP, Weiner DJ, Yu Q-C and Wilson JM. 2001. Filovirus-pseudotyped lentiviral vector can efficiently and stably transducer airway epithelia in vivo. Nat. Biotechnol, 19:225-230.
- Watson DJ, Kobinger GP, Passini MA, Wilson JM and Wolfe JH. 2002. Targeted transduction patterns in the mouse brain by lentivirus vectors pseudotyped with VSV, Ebola, Mokola, LCMV, or MuLV envelope proteins. Mol. Therapy, 5:528-537.
- MacKenzie TC, Kobinger GP, Kootstra NA, Radu A, Sena-Esteves M, Bouchard S, Wilson JM, Verma IM and Flake AW. 2002. Efficient transduction of liver and muscle after in utero injection of lentiviral vectors with different pseudotypes. Mol. Therapy, 6:349-358.