Kathryn K. Harris

The Escherichia coli helicase, YoaA, and DNA polymerase III subunit, χ, form a complex (YoaA-χ) that promotes tolerance to the DNA chain-terminator 3ˈ-azidothymidine (AZT). Single-stranded DNA binding protein (SSB), which accumulates at stalled replication forks, also contributes to AZT tolerance through interactions with χ. Here we show that in vitro, χ mediates interactions between YoaA and SSB that modulate helicase activity in a substrate-specific manner with little effect on overhang DNA but inhibiting unwinding of forked DNA. SSB similarly affects the activity of the YoaA paralog, DinG. Single-molecule experiments show that SSB translocates with YoaA-χ, increasing both the lifetime and frequency of SSB binding events. Mutational analyses show that χ binds at the back of YoaA relative to the direction of translocation supporting a model in which YoaA-χ pulls SSB along DNA as it translocates. To our knowledge, this is the first demonstration of a mechanoenzyme pulling SSB along ssDNA.

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 10¹² viral particles/mL of exceptional purity for in vivo use.

Recombinant adeno-associated viruses (rAAVs) have become one of the leading gene therapies for treating a variety of diseases. One factor contributing to rAAVs’ success is the fact that a wide variety of tissue types can be transduced by different serotypes. However, one commonality amongst most serotypes is the high propensity for liver transduction when rAAVs are administered peripherally. One of the few exceptions is the naturally occurring clade F AAV hematopoietic stem cell 16 (AAVHSC16). AAVHSC16 represents an interesting capsid in that it shows minimal liver transduction when injected peripherally. For capsids other than AAVHSC16, targeting non-liver tissues via peripheral AAV injection represents a challenge due to the high liver transduction. Thus, there is a demand for liver-de-targeted rAAV vectors. The rational design of rAAV capsids relies on current knowledge to design improved capsids and represents one means of developing capsids with reduced liver transduction. Here, we utilized data from the AAVHSC16 capsid to rationally design four non-clade F rAAV capsids that result in reduced liver transduction following peripheral injection.