Helper Dependent Adenovirus

One of the major drawbacks of replication incompetent adenoviruses (also commonly referred to as “First Generation” adenoviruses) is that the deletion of the E1 gene is not enough to totally obliterate all viral gene activation.

“Leaky” viral gene expression from the vector itself results in the generation of anti-Ad cytotoxic T-lymphocytes. This kind of immune response not only impacts the duration of transgene expression in vivo but it also results in significant acute inflammatory reactions in the host.

Eliminating more of the viral genome can serve to reduce Ad-induced toxicity induced from this low level viral (early and late) gene expression. However, aside from removal of the E3 gene which is dispensable to growth, further deletions of viral genes relies on the supplementation of those viral gene products via the complementing cell line. While this does serve to improve on the longevity of transgene expression, the development of such continuous complementing cell lines is time consuming and difficult.

Helper dependent (HD) adenovectors have incurred deletion of all viral genes. They contain only the cis-acting elements. This encompasses the left and right ITR sequences (critical for DNA replication) as well as the packaging region (required for encapsidation of the vector genome). This amounts to retaining ~600bp of the adenoviral genome. The remaining intervening section is filled with non-coding “stuffer DNA”. Such elimination of viral DNA ensures that no viral genes are present for expression from the HD backbone itself, hence offering the potential for reduced toxicity and prolonged transgene expression. In addition, it also allows for greatly increased cloning capacity of such vectors to ~37 kb. This is an important factor since the cloning capacity can be a significant limitation for many other delivery systems.

Being devoid of all viral genes, HD vectors have an absolute requirement for all viral gene products (aside from E3) for their propagation. These gene products can be supplied in trans most conveniently via a “Helper virus”.

A Helper virus is essentially a first generation virus with the capacity to dominate over the HD vector in tissue culture propagation. Therefore it is critical to incorporate a system which will incapacitate the Helper virus to allow the HD virus to become the dominant viral population. The Cre/loxP system is one method that can achieve this goal.

This system entails two components. One is the incorporation of the loxP sites which flank the packaging signal of the Helper virus. The second is the development of Cre recombinase transformed 293 cell line (293Cre4).

The use of both components allow for the initial growth of the Helper viral DNA to replicate and provide the necessary viral gene products in trans for propagation of the co-infected recombinant HD (rHD) vector.

The Cre recombinase supplied by the 293Cre4 cells line then acts to excise the packaging signal through recombination between the flanking loxP sites, rendering the helper genome unpackageable.

The HD genome, on the other hand is unaffected by the Cre recombinase and is free to be packaged into the preformed capsids. The titre of the HD vector is increased by serial coinfection of 293cre4 cells with the HD vector and the Helper virus. Following sufficient level of expansion of the rHD virus, the final step requires CsCl ultracentrifugation for purification.

Each Helper Dependent Adenovirus Kit Contains:
Catalogue # Product
PD-01-70 Helper Dependent Adenovirus Vector Kit K
  • Approximately 106 293Cre4 cells
  • Approximately 107 PFU adeno helper virus H14
  • 10 micrograms of plasmid pC4HSUgfp
  • 10 micrograms of plasmid pC4HSU

Helper Dependent adenovirus vectors are covered by US Patent nos. 7,045,347; 6,730,507; 6,566,128; 6,080,569; 5,919,676

Ready to proceed with an order? Contact customer service for pricing and availability of all items.

References

Parks, R.J., L. Chen, M. Anton, U. Sankar, M.A. Rudnick, and F.L. Graham. A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc. Natl. Acad. Sci. USA 93: 13565-13570, 1996.

Parks, R.J., and F.L. Graham. A helper dependent system for adenovirus vector production helps define a lower limit for efficient DNA packaging. J. Virol. 71:3293-3298, 1997.

Kochanek, S., P.R. Clemens, K. Mitani, H-H Chen, S. Chan, and C.T. Caskey. A new adenoviral vector: replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and b-galactosidase. Proc. Natl. Acad. Sci. USA 93: 5731-58736, 1996.

Sandig, V., R. Youil, A.J. Bett, L.L. Franlin, M. Oshima, D. Maione, F. Wang, M.L. Metzker, R.Savino, and C.T. Caskey. Optimization of the helper-dependent adenovirus system for production and potency in vivo. P.N.A.S. 97:1002-1007, 2000.

Gorziglia, M.I., C. Lapcevich, S. Roy, Q. Kang, M. Kadan, V. We, P. Pechan, and M. Kaleko. Generation of an adenovirus vector lacking E1, E2a, E3 and all of E4 except open reading frame 3. J. Virol. 73: 6048-6055, 1999.

O’Neal, W.K., H. Zhou, N. Morral, C. Langston, R.J. Parks, F.L. Graham, S. Kochanek, and A.L. Beaudet. Toxicity associated with repeated administration of first-generation adenovirus vectors does not occur with a helper-dependent vector. Molecular Medicine 6: 179-195, 2000.

Amalfitano, A, M.A. Hauser, H. Hu, D. Serra, C.R. Begy, and J.S. Chamberlain. Production and characterization of improved adenovirus vectors with the E1, E2b, and E3 genes deleted. J. Virol. 72: 926-933, 1998.

Morsy, M.A., M. Gu, S. Motzel, J. Zhao, J. Li, Q. Su, H. Allen, L. Franlin, R.J. Parks, F.L. Graham, S. Kochanek, A.J. Bett, and C.T. Caskey. An adenoviral vector deleted for all viral coding sequences results in enhanced safety and extended expression of a leptin transgene. Proc. Natl. Acad. Sci. USA 95: 7866-7871, 1998.

Hartigan-O’Connor, D., A. Amalfitano, and J. Chamberlain. Improved production of gutted adenovirus in cells expressing adenovirus preterminal protein and DNA polymerase. J. Virol. 73: 7835-7841, 1999