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Abstract

Adeno-associated virus (AAV) is a member of the Parvoviridae family with a non-enveloped and icosahedral capsid structure. Recombinant AAVs (rAAV) are non-pathogenic with the low immunogenicity and broad cell/tissue tropism, thus AAV is wildly used as a gene therapy tool. Currently, many clinical trials involving AAV vectors are ongoing and the marketed drug Glybera® was approved in Europe in 2012, followed by the Luxturna by FDA approval in 2017.

However, the limitation of this vector is the poor AAV transduction efficiency. To reveal the regulation of the host factors in AAV2 transduction, an RNAi screen was performed previously to identify host proteins interfering with AAV2 transduction. Sae2 and Ubc9, which are key enzymes of the SUMOylation pathway, were identified as restriction factors of AAV infection. Similar to the ubiquitination, SUMOylation is a post-translational protein modification catalyzed by an E1 activating enzyme (consisting of Sae1 and Sae2) and an E2 conjugating enzyme (Ubc9).

Further investigations in this thesis confirmed that the impairment of SUMOylation via Sae2 or Ubc9 knockdown resulted in a higher AAV transduction efficiency, and AAV2 infection can also enhance the total SUMOylation activity of host cells. Moreover, SUMOylation affects the transduction of AAV vectors with single stranded DNA (ssAAV) and self-complementary DNA (scAAV).

To determine the target of SUMOylation in the AAV life cycle, a pull-down assay was performed for AAV2 particles indicating that the AAV2 capsid protein is SUMOylated. Site-directed mutagenesis and modified virus transduction shows involvement of K142/143 and K169 of VP2 in capsid SUMOylation. In addition, the data suggests that the N-terminus of VP2 needs to be free, as addition of protein tags such as GFP or HA abolishes SUMOylation. This indicates that the AAV2 capsid SUMOylation has VP2 spatial structure requirements.

Moreover, the observed increased AAV2 transduction in a Daxx knockout cell line indicates that Daxx is another restriction factor of AAV. Compared with wild type HeLa cell line, the enhancing affects after Ubc9 knockdown in Daxx knockout cell line was dramatically reduced, which means the Daxx protein and SUMOylation may work in the same or overlapping pathways. Thence, not only the AAV2 capsid protein, but also SUMOylated Daxx could regulate AAV transduction.

AAV2 subcellular trafficking involves multiple steps. AAV2 vector DNA in the cell membrane and cytoplasmic fractions are not altered after Sae2 knockdown indicating that impairment of the SUMOylation-pathway cannot affect AAV binding and the intracellular transport and therefore have no influence on AAV transduction. Other than this, there are two possibilities for SUMOylated AAV2 particles to accumulate in the nucleus: SUMOylation pathway could restrict the number of particles that reach the nucleus and undergo transduction; Alternatively, SUMOylation could not affect the amount of AAV2 particles but instead enhance AAV transduction by promoting AAV uncoating and ssDNA release in the nucleus.