539. Screening for Recombinant Adeno-Associated Viral Vectors That Selectively Transduce Hepatitis B Virus Infected Cells
Year: 2016
Type: article
Abstract: Hepatitis B is a disease caused by infection with the hepatitis B virus (HBV), a small enveloped DNA virus which belongs to the Hepadnaviridae family. Even though a safe and effective vaccine is available, HBV represents the ninth most common cause of death worldwide with an estimated 350 million chronically infected individuals. HBV infection can cause cirrhosis and hepatocellular carcinoma and currently there are 7 approved drugs for the treatment of HBV infection in the USA. These drugs decrease the risk of liver damage from HBV by slowing but not eliminating viral replication in most patients, possibly related to the persistence of integrated HBV genomes. Gene therapy using recombinant adeno-associated viral (rAAV) vectors constitute a promising tool to combat HBV infection as rAAV vectors have the ability to transduce and establish long-term and stable transgene expression in liver cells. Preclinical studies have successfully utilized rAAV vectors to combat viral infections after delivering short-hairpin RNA (shRNA) expression cassettes for knocking down viral coding or host genes required for viral replication and spread. Because promiscuous targeting of host cellular genes in the majority of non-infected cells may be detrimental, specific targeting of HBV-infected cells adds a new layer of potential therapeutic targets and safety. Thus we set out to identify new AAV capsids that selectively transduce HBV-infected cells. To do this, we created new AAV shuffled capsid libraries and used these for multiple rounds of infection in HBV-infected hepatoma cells. Each library is co-infected with wild-type Adenovirus-5 (helper virus) allowing the rAAV genomes to replicate in the HBV-infected cells. Isolation of replicating rAAV over successive passages in cultured cells allows for a more stringent selection as all steps in AAV transduction must occur. We have varied the screens using various multiplicity of infections (MOIs) of the library and/or utilized a pre-clearing step using a HBV negative hepatoma cell line. Several selected capsids derived from multiple parental capsids were vectorized. One capsid (AAV-GK4) was found to transduce the infected cells approximately 6 times more robustly than uninfected cells (using an MOI of 100 and 1,000). Interestingly, the same result was observed with AAV-DJ, a variant previously selected on human hepatoma cells. Additional screens and candidates are pending further evaluation. The possibility of finding new rAAV capsids with an increased transduction efficiency and selective tropism for HBV-infected cells will provide a reagent or therapeutic that can deliver a transgene that will allow one to selectively modify or potentially kill infected but not the uninfected cells contained within the liver.
Source: Molecular Therapy
Authors: Gustavo de Alencastro, Katja Pekrun, Mark Kay
Institution: Stanford University
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $4,500
OpenAlex ID: W2561412683