NextGen, GenX, and Opt AAV3 Vectors

Recombinant AAV vectors have been, or are currently being, used in 176 Phase I/II/III trials in humans and have shown clinical efficacy in a wide variety of human diseases. One AAV vector was approved as a drug, designated as Alipogene Tiparvovec and marketed under the trade name Glybera® in Europe in 2012. In 2017, a second AAV vector, Voretigene Neparvovec (Luxturna®), was approved in the USA. Despite these remarkable achievements, the full potential of AAV vectors, when composed of the naturally occurring, wild-type (WT) capsids and genomes, is unlikely to be realized for the following reasons:

  • AAV capsid is targeted for degradation by the host cell enzymes, which leads to host immune responses to capsid proteins
  • The single-stranded AAV genome is transcriptionally-inactive, which impacts on the expression levels of transgenes

... aaVective has overcome both limitations.

NextGen

aaVective has developed NextGen AAV3 vectors, illustrated here, which decrease the natural propensity of host immune response.

Surface-exposed tyrosine (Y), serine (S), threonine (T), and lysine (K) residues in the first generation AAV3 vectors are mutagenized to phenylalanine (F), valine (V), and arginine (R) residues, respectively, in the NextGen AAV3 vectors that are more efficient and less immunogenic.

GenX

aaVective has overcome the limitation of the wild-type single-stranded AAV genome with the development of GenX AAV3 vectors,  depicted below.

Specific DNA  sequences in the inverted terminal repeats in the ssAAV genome (depicted in red) are replaced by a substitute sequence (depicted in green) in the GenX AAV vectors that mediate more efficient transgene expression.

Opt

aaVective has also demonstrated that combining NextGen capsids with GenX genomes leads to the generation of optimized (Opt) AAV vectors, shown schematically below, that are more efficient at further reduced doses.

Encapsidation of the GenX genome into the NextGen capsid leads to the generation of more efficient optimized (Opt) AAV vectors at further reduced doses.

aaVective’s NextGen, GenX, and Opt AAV3 vectors have been shown to be highly efficient in human liver cells in vitro, and in mouse xenograft models in vivo. aaVective’s NextGen AAV3 vectors have also been evaluated for their safety and efficacy in “humanized” mice and in non-human primates, and shown to perform significantly better than first generation AAV5 and AAV8 vectors, the two other serotype vectors currently being used by other AAV hemophilia gene therapy companies.

Relevant aaVective Publications

NextGen AAV vectors

  • Zhong, W. Zhao, J. Wu, B. Li, B. Li, S. Zolotukhin, L. Govindasamy, M. Agbandge-McKenna, and A. Srivastava. A dual role of EGFR protein tyrosine kinase signaling in ubiquitination of AAV2 capsids and viral second-strand DNA synthesis. Mol. Therapy, 15: 1323-1330, 2007.
  • Zhong, B. Li, C.S. Mah, L. Govindasamy, M. Agbandje-McKenna, M.A. Cooper, R.W. Herzog, I. Zolotukhin, K.H. Warrington, Jr., K.A. Weigel-Van Aken, J.A. Hobbs, S. Zolotukhin, N. Muzyczka, and A. Srivastava. Next generation of adeno-associated virus 2 vectors: Point mutations in tyrosines lead to high-efficiency transduction at reduced doses. Proc. Natl. Acad. Sci., USA, 105: 7827-7832, 2008.
  • Markusic, R.W. Herzog, G. Aslanidi, B. Hoffman, B. Li, M. Li, G.R. Jayandharan, C. Ling, I. Zolotukhin, W. Ma, S. Zolotukhin, A. Srivastava, and L. Zhong. High-efficiency transduction and correction of murine hemophilia B using AAV2 vectors devoid of multiple surface-exposed tyrosines. Mol. Therapy, 18: 2048-2056, 2010.
  • G.V. Aslanidi, A.E. Rivers, L. Ortiz, L. Govindasamy, C. Ling, G.R. Jayandharan, S. Zolotukhin, M. Agbandje-McKenna, and A. Srivastava. High-efficiency transduction of human monocyte-derived dendritic cells by capsid-modified recombinant AAV2 vectors. Vaccine, 30: 3908-3917, 2012.
  • A.T. Martino, E. Basner-Tschakarjan, D.M. Markusic, J. Finn, C. Hinderer, S. Zhou, D.A. Ostrov, A. Srivastava, H.C.J. Ertl, C. Terhorst, K.A. High, F. Mingozzi, and R.W. Herzog. Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells.  Blood, 121: 2224-33, 2013.
  • G.V. Aslanidi, A.E. Rivers, L. Ortiz, C. Ling, L. Song, L. Govindasamy, M. Tan, M. Agbandje-McKenna, and A. Srivastava. Optimization of recombinant AAV2 vectors for gene therapy: The final threshold? PLoS One, 8(3): e59142, 2013.
  • Ling, Y. Wang, Y. Zhang, A. Ejjigani, Z. Yin, Y. Lu, L. Wang, M. Wang, J. Li, Z. Hu, G.V. Aslanidi, L. Zhong, G. Gao, A. Srivastava, and C. Ling. Selective in vivo targeting of human liver tumors by optimized recombinant AAV3 vectors in a murine xenograft model. Human Gene Therapy, 25: 1023-1034, 2014.
  • Li, C. Ling, L. Zhong, M. Li, Q. Su R. He, Q. Tang, D.L. Greiner, L.D. Shultz, M.A. Brehm, T.R. Flotte, C. Mueller, A. Srivastava, and G. Gao. Efficient and targeted transduction of nonhuman primate liver with systemically delivered optimized AAV3B vectors. Mol. Therapy, 23: 1867-1876, 2015.
  • K. Vercauteren, B.E. Hoffman, I. Zolotukhin, J.W.  Xiao, E. Basner-Tshakarjan, K.A. High, H.C.J. Ertl, C.M. Rice, A. Srivastava, Y.P. de Jong, and R.W. Herzog. Superior in vivo transduction of human hepatocytes using engineered AAV3 capsid. Mol. Therapy, 24: 1042-1049, 2016.

GenX AAV vectors

  • K.Y. Qing, X.-S. Wang, D.M. Kube, S. Ponnazhagan, A. Bajpai and A. Srivastava.  Role of tyrosine phosphorylation of a cellular protein in adeno-associated virus 2-mediated transgene expression. Proc. Natl. Acad. Sci., USA, 94: 10879-10884, 1997.
  • X.-S. Wang, K.Y. Qing, S. Ponnazhagan and A. Srivastava. Adeno-associated virus 2 DNA replication in vivo: Mutation analyses of the D-sequence in viral inverted terminal repeats. J. Virology, 71: 3077-3082, 1997.
  • Mah, K.Y. Qing, B. Khuntirat, S. Ponnazhagan, X.-S. Wang, D.M. Kube, M.C. Yoder and A. Srivastava. Adeno-associated virus 2-mediated gene transfer: Role of epidermal growth factor receptor protein tyrosine kinase in transgene expression. J. Virology, 72: 9835-9843, 1998.
  • K.Y. Qing, B. Khuntirat, C. Mah, D.M. Kube, X.-S. Wang, S. Ponnazhagan, S.Z. Zhou, V.J. Dwarki, M.C. Yoder and A. Srivastava. Adeno-associated virus type 2-mediated gene transfer: Correlation of tyrosine phosphorylation of the cellular single-stranded D sequence-binding protein with transgene expression in human cells in vitro and murine tissues in vivo. J. Virology, 72: 1593-1599, 1998.
  • K.Y. Qing, J. Hansen, K.A. Weigel-Kelley, M.Q. Tan, S.Z. Zhou and A. Srivastava.  Adeno-associated virus 2-mediated gene transfer:  Role of cellular FKBP52 protein in transgene expression. J. Virology, 75: 8968-8976, 2001.
  • K.Y. Qing, W. Li, L. Zhong, M.Q. Tan, J. Hansen, K.A. Weigel-Kelley, L. Chen, M.C. Yoder and A. Srivastava.  Adeno-associated virus 2-mediated gene transfer:  Role of T-cell protein tyrosine phosphatase in transgene expression in established cell lines in vitro and transgenic mice in vivo. J. Virol., 77: 2741-2746, 2003.
  • Zhong, L.Y. Chen, Y. Li, K.Y. Qing, K.A. Weigel-Kelley, R.J. Chan, M.C. Yoder, and A. Srivastava. Self-complementary adeno-associated virus 2 (AAV)-T cell protein tyrosine phosphatase vectors as helper-viruses to improve transduction efficiency of conventional single-stranded AAV vectors in vitro and in vivo. Mol. Therapy, 10: 950-957, 2004.
  • Zhao, L. Zhong, J. Wu, L. Chen, K. Qing, K.A. Weigel-Kelley, S.H. Larsen, W. Shou, K.H. Warrington, Jr., and A. Srivastava. Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors. Virology, 353: 283-293, 2006.
  • Zhao, J. Wu, L. Zhong, and A. Srivastava. Adeno-associated virus 2-mediated gene transfer: Identification of a cellular serine/threonine protein phosphatase involved in augmenting vector transduction efficiency. Gene Therapy, 14: 545-550, 2007.
  • Zhong, X. Zhou, Y. Li, K.Y. Qing, X. Xiao, R.J. Samulski, and A. Srivastava. Single-polarity recombinant adeno-associated virus 2 vector-mediated transgene expression in vitro and in vivo: Mechanism of transduction. Mol. Therapy, 16: 290-295, 2008.
  • G.R. Jayandharan, L. Zhong, B.K. Sack, A.E. Rivers, M. Li, B. Li, R.W. Herzog, and A. Srivastava. Optimized adeno-associated virus (AAV)-protein phosphatase-5 helper viruses for efficient liver transduction by single-stranded AAV vectors: Therapeutic expression of Factor IX at reduced vector doses. Hum. Gene Therapy, 21: 271-283, 2010.
  • Cheng, C. Ling, Y. Dai, L.G. Glushakova, Y. Lu, S.W.Y. Gee, K.E. McGoogan, G.V. Aslanidi, M. Park, P.W. Stacpoole, D. Siemann, C. Liu, A. Srivastava, and C. Ling. Development of optimized AAV3 serotype vectors: Mechanism of high-efficiency transduction of human liver cancer cells. Gene Therapy, 19: 375-384, 2012.
  • Ling, Y. Wang, Y. Lu, L. Wang, G.R. Jayandharan, G.V. Aslanidi, B. Li, B. Cheng, W. Ma, T. Lentz, C. Ling, X. Xiao, R.J. Samulski, N. Muzyczka, and A. Srivastava. Enhanced transgene expression from recombinant single-stranded AAV vectors in human cell lines in vitro and in murine hepatocytes in vivo. J. Virology, 89: 952-961, 2015.
  • Li, W. Ma, G.V. Aslanidi, C. Ling, K.V. Vliet, L.-y. Huang, M. Agbandje-McKenna, A. Srivastava, and G.A. Aslanidi. Site-directed mutagenesis of surface-exposed lysine residues leads to high-efficiency transduction by recombinant AAV2, but not AAV8 vectors. Hum. Gene Therapy Methods, 26: 211-220, 2015.

Opt AAV Vectors

  • Ling, B. Li, W. Ma, and A. Srivastava. Development optimized AAV serotype vectors for high-efficiency transduction at further reduced doses. Hum. Gene Therapy Methods, 27: 143-149, 2016.

Supporting publications

  • Ponnazhagan, P. Mukherjee, M.C. Yoder, X.-S. Wang, S.Z. Zhou, J. Kaplan, S. Wadsworth and A. Srivastava. Adeno-associated virus 2-mediated gene transfer in vivo: Organ-tropism and expression of transduced sequences in mice. Gene, 190: 203-210, 1997.
  • K.Y. Qing, C. Mah, J. Hansen, S.Z. Zhou, V.J. Dwarki and A. Srivastava. Human fibroblast growth factor receptor 1 is a co-receptor for infection by adeno-associated virus 2. Nature Medicine, 5: 71-77, 1999.
  • Zhong, W. Li, Z, Yang, L.Y. Chen, Y. Li, K.Y. Qing, K.A. Weigel-Kelley, M.C. Yoder, W. Shou and A. Srivastava. Improved transduction of primary murine hepatocytes by recombinant adeno-associated virus 2 vectors in vivo. Gene Therapy, 11: 1165-1169, 2004.
  • Zhong, K.Y. Qing, Y. Si, L. Chen, M.Q. Tan and A. Srivastava. Heat-shock treatment-mediated increase in transduction by adeno-associated virus 2-vectors is independent of the cellular heat-shock protein 90. J. Biol. Chem., 279: 12714-12723, 2004.
  • L.G. Glushakova, M.J. Lisankie, E.B. Eruslanov, C. Ojano-Dirain, I. Zolotukhin, L. Zhong, C. Liu, A. Srivastava, and P.W. Stacpoole. AAV3-mediated transfer and expression of the pyruvate dehydrogenase E1a subunit gene causes metabolic remodeling and apoptosis in human liver cancer cells. Mol. Genet. Metabol., 98: 289-299, 2009.
  • Ling, Y. Lu, J. Kalsi, G.R. Jayandharan, B. Li, W. Ma, B. Cheng, S. Gee, K. McGoogan, L. Zhong, L. Govindasamy, M. Agbandje-McKenna, and A. Srivastava. Hepatocyte growth factor receptor is a cellular coreceptor for adeno-associated virus 3. Hum. Gene Therapy, 21: 1741-1747, 2010.
  • Ling, Y. Lu, B. Cheng, K.E. McGoogan, S.W.Y. Gee, W. Ma, B. Li, G.V. Aslanidi, and A. Srivastava. High-efficiency transduction of liver cancer cells by recombinant adeno-associated virus serotype 3 vectors. J. Vis. Exp., 49. Pii: 2538, doi: 10.3791/2538, 2011.
  • Ling, Y. Wang, Y. Lu, L. Wang, G.R. Jayandharan, G.V. Aslanidi, B. Li, B. Cheng, W. Ma, T. Lentz, C. Ling, X. Xiao, R.J. Samulski, N. Muzyczka, and A. Srivastava. The adeno-associated virus genome packaging puzzle. J. Mol. Genet. Medicine, 9: 3, 1000178, 2015.
  • C. Ling, Z. Yin, J. Li, D. Zhang, G. Aslanidi, and A. Srivastava. Strategies to generate high-titer, high-potency recombinant AAV3 serotype vectors. Mol. Ther. Methods & Clin. Dev., 3:16029, 2016.