Capsid-modified, RAAV3 vector compositions and uses in gene therapy of human liver cancer
A composition and carrier technology, applied in the fields of molecular biology and virology, can solve problems such as inapplicability and achieve the effect of improving efficiency
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Embodiment 1-
[0216] Example 1 - Next Generation rAAV2 Vectors: Point Mutation in Tyrosine Leads to Efficient Transduction at Lower Doses
[0217] This example demonstrates that mutation of surface-exposed tyrosine residues on the AAV2 capsid bypasses the ubiquitination step, thereby avoiding proteasome-mediated degradation, and results in the loss of these vectors in human cells in vitro and in mouse cells in vivo. Efficient transduction in hepatocytes, resulting in the production of therapeutic levels of human coagulation factors at reduced vector doses. The observed increased transduction efficiency of tyrosine mutant vectors is due to lack of ubiquitination and improved intracellular trafficking to the nucleus. In addition to yielding a deep understanding of the role of tyrosine phosphorylation of the AAV2 capsid in multiple steps of the AAV2 life cycle, these studies have led to the development of new AAV2 vectors capable of efficient transduction at lower doses.
[0218] Materials an...
Embodiment 2-r
[0244] Example 2 - Activation of NF-κB pathway by rAAV vector
[0245] Since in silico analysis using the human transcription factor database demonstrated that there are multiple binding sites for NF-κB (central regulator of cellular immune and inflammatory responses) in the adeno-associated virus (AAV) genome, this example studies Whether AAV utilizes NF-κB during its life cycle. Small molecule modulators of NF-κB were used in HeLa cells transduced with recombinant AAV vectors. VP16, an NF-κB activator, which enhanced AAV vector-mediated transgene expression up to 25-fold. Of the two NF-κB inhibitors, (Bay11), which blocks both the canonical and non-canonical NF-κB pathways, completely abolished transgene expression, whereas pyrrolidonedithiocarbamate (PDTC), which interfered with the canonical NF-κB pathway, ) had no effect on transgene expression. Western blot analysis confirmed the enrichment of the nuclear p52 protein component of the non-canonical NF-κB pathway in the...
Embodiment 3
[0270] Example 3 - Development of optimized AAV3 serotype vectors
[0271] Adeno-associated virus 2 (AAV2) is a non-pathogenic human parvovirus that contains a single-stranded DNA genome and has broad tissue tropism across species barriers (Muzyczka, 1992). Recombinant AAV2 vectors have gained attention as a promising vector system for potential gene therapy of various human diseases and are currently used in several gene therapy clinical trials (Daya and Berns, 2008). Recently, various additional AAV serotypes have been isolated and found to efficiently transduce specific cell types (Muramatsu et al., 1996; Chiorini et al., 1997; Chiorini et al., 1999; Rutledge et al., 1998; GaoGP et al., 2002; Vandenberghe et al. et al., 2004). Although the multiple steps in the AAV2 life cycle are fairly well understood (Summerford and Samulski 1998; Qing et al., 1999; Summerford et al., 1999; Hansen et al., 2000; Hansen et al., 2001; Sanlioglu et al., 2000; Douar et al., 2001; Zhao et al...
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