UK COBRE: Viral Production Core

Viral Production Core

The use of viral vectors in biomedical and translation research has increasingly become the method of choice for expression of genes in difficult to transfect cells, for long-term expression of genes of interest, or for reducing the expression of a particular gene of interest. Viral vectors are useful for expression of recombinant proteins in both cell lines and whole animals. It is clearly the method of choice to introduce genes and silencing RNAs into post mitotic cells, and viral vectors provide a rapid and targeted method for gene delivery. The most commonly used viral vectors are lentivirus vectors, adenovirus vectors, and adeno-associated virus vectors. Each has its associated advantages and disadvantages. For example lentivirus is most useful for non-dividing CNS cells. The onset of lentivirus expression is rapid, but viral titers are generally low. Adeno-associated virus expression with its many serotypes can be used in a larger variety of tissues. It takes several days to reach its maximal level, but titers of virus are generally high. Adenovirus is easier to generate, but does not produce long-term expression. Thus it is not surprising that different labs use different viral vectors depending on their particular experimental system. These viral vectors have in common the fact that production of the virus is generally laborious, requiring a skilled and experienced individual and sterile and safe working conditions.

There are a number of projects associated with our COBRE grant on the Molecular Basis of Human Disease that use viral vectors and we expect these to expand in the near future. Examples are listed below, and it is worth noting that this list include the projects of all the current project PIs:

Douglas Andres, COBRE Mentor
Project Title: Physiological function of Ras family GTPases
Dr. Andres will infect primary cardiac myocytes and neurons cells with adenoviral vectors expressing specific proteins of interest (whole proteins, functional domains or mutated forms) or reporter genes controlled by a promoter sequence of interest. Lentiviral vectors will also be utilized in primary hippocampal neurons to knockdown specific proteins by sh/miRNA expression.

Matthew Gentry, COBRE Project PI
Project Title: Role of AMP-activated protein kinase in Lafora and Wolff-Parkinson-White disease
Dr. Gentry will utilize lentivirus expression vectors to deliver wild type and mutant proteins, both full length as well as specific domains of interest, into mouse embryonic fibroblasts as well as primary neurons. In addition, he will use lentiviral vectors to knockdown specific proteins of interest by sh/miRNA expression in mouse embryonic fibroblasts and primary neurons.

Louis Hersh, COBRE PI
Project Title: Peripheral expression of a peptidase to treat Alzheimer’s disease
Dr. Hersh has been using both lentivirus and adeno-associated virus to express the peptidase neprilysin as a method to clear amyloid from the brain. This translational project is moving toward clinical trials.

Christopher M. Norris, Former COBRE Pilot Grant PI
Project Title: Calcineurin and inflammatory signaling processes in aging and Alzheimer’s disease
Adenoviruses are used to manipulate calcineurin signaling pathways in primary cell cultures and adeno-associated viruses are used to target calcineurin signaling in astrocytes of intact APP/PS1 mice and aged rats.

Sabire Ozcan, Former COBRE Mentor
Project Title: Histone Acetylation and Insulin Gene Expression
This project focuses on insulin production and secretion from pancreatic beta cells. Pancreatic beta cell lines and pancreatic islets are very difficult to transfect using conventional transfection methods. To achieve reasonable transfection, we have to use adenoviral as well as lentiviral gene transfer.

Francesc Marti, COBRE Project PI
Project Title: Mechanisms of Peripheral Regulatory T Cell Generation
Dr. Marti will infect primary T cells with adenoviral vectors expressing specific proteins of interest (whole proteins, functional domains or mutated forms) or reporter genes controlled by a promoter sequence of interest. Lentiviral vectors will be also be utilized in primary T cells to knockdown specific proteins by sh/miRNA expression.

Christian Paumi, COBRE Project PI
Project Title: Negative Regulation of the Yeast MRP, Ycf1p, and Human MRPs by CKII
In this project Dr. Paumi will use adeno-associated virus to make MCF7 breast cancer cells that stably overexpress MRP6 (ABCC6), CK2alpha, and CK2alpha. These cells will then be used to determine the role of CK2 in MRP-mediated multidrug resistance. Further, we will use adeno-associated virus to transduce shRNA plasmids into MCF7 cells (WT and MRP1 and MRP6 overexpressing cells) resulting in CK2 knock-downs.

M. Paul Murphy, Former COBRE Project PI
Project Title: Cellular Nucleic Acid Binding Protein (CNBP) in Aging and Disease
In this project the hypothesis will be tested that CNBP is involved in the development of neurodegenerative and age related disease in vivo. AAV will be used to overexpress and knock down CNBP in a well-characterized mouse model of Aβ pathogenesis. Both brain and skeletal muscle will be studies using both CNBP and anti-CNBP shRNAs.

Stefan Stamm, Former COBRE Pilot Grant PI
Project Title: Regulation of alternative pre-mRNA processing by small nucleolar RNAs
Dr. Stamm will use lentivirus to deliver snoRNAs into neurons. It will be tested whether the snoRNAs in these lentiviruses are processed properly and can substitute the loss of snoRNA expression seen in patients with Prader-Willi syndrome. The processing of the snoRNAs will be determined by RNAse protection assays and compared with the situation in brain. If we encounter processing problems with lentiviruses, we will use adenovirus delivery as an alternative.

Haining Zhu, Core Director and Former COBRE Project PI
Project Title: Degradation and aggregation of mutant SOD1 related with familial Lou Gehrig’s disease
Dr. Zhu will use adeno-associated virus for infecting primary neurons in his studies of ALS, particularly for studying the degradation and aggregation processes of mutant SOD1 related to familial ALS. AAV vectors will be utilized in primary neurons to express or knock down proteins to examine the role of axonal transport and autophagy activation in the homeostasis of the disease-related mutant SOD1 protein.

Lentivirus vectors will be made using a four plasmid transfection system (Dull, T., Zufferey, R., Kelly, M., Mandel, R. J., Nguyen, M., Trono, D. & Naldini, L. (1998) J Virol 72, 8463-71) from which high vector titers can be achieved. The use of this four-plasmid system in which all HIV accessory genes have been removed, drastically reduces the risk of generation of a replication competent lentivirus. To further ensure the safety of our vectors the SIN system is used (Miyoshi et al. (1998) J Virol 72, 8150-7) which incorporates a deletion in the 3’ LTR ensuring that no replicating virus can arise since no intact LTR is available to drive expression of viral RNA. The lentiviral vectors also contain additional cis acting Woodchuck hepatitis virus posttranscriptional regulatory element and central poly-purine track elements to further improve their efficiency. Lastly the vectors are pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G, in place of the natural HIV envelope glycoprotein) to allow the vector entry into a wide range of cell types.

Adeno-associated viruses are currently being produced in Dr. Telling’s lab using the insect cell-baculovirus based packaging system developed by Dr. Robert Kotin (NIH). The insect packaging approach is a multi-step system designed to produce replication deficient rAAV. Briefly, a transgene cassette is cloned into a pFastBac plasmid containing inverted terminal repeats (ITR’s). Concurrently, two more plasmids are produced containing the Rep and Cap ORF’s of wild type AAV. All three plasmids are then transformed separately into MAX Efficiency® DH10Bac to produce bacmid molecules (Bac-to-Bac, Invitrogen). Each bacmid is transfected into Sf9 insect cells to produce baculovirus. The baculovirus containing the three respective constructs is titered and co-transduced back into Sf9 cells for propagation. The Rep and Cap helper genes are supplied separately in trans via packaged baculoviruses.

For the production of adenoviruses, genes of interest will be subcloned into the adenoviral vector (pAdTrackCMV, obtained from Dr. Vogelstein, John Hopkins University) behind the CMV promoter (Luo et al., Nat Protoc (2007); 2(5):1236-47). The constructs will be transformed into bacteria together with pAdEASY (contains the adenoviral genome without the E1 gene) and selected for recombinant plasmids containing the gene of interest in the adenoviral genome. The resultant recombinant plasmid will be used to transfect HEK 293 cells that contain the adenoviral E1 gene required for replication of the virus. Thus, the recombinant adenovirus can only be amplified in HEK 293 cells. After amplification and purification, the recombinant adenoviruses can be used to infect a wide range of cultured cell lines to allow over-expression of genes of interest.