Application of in-vitro-cultured primary hepatocytes to evaluate species translatability and AAV transduction mechanisms of action.

Recombinant adeno-associated virus (AAV) is an effective platform for therapeutic gene transfer; however, tissue-tropism differences between species are a challenge for successful translation of preclinical results to humans. We evaluated the use of in vitro primary hepatocyte cultures to predict in vivo liver-directed AAV expression in different species. We assessed whether in vitro AAV transduction assays in cultured primary hepatocytes from mice, nonhuman primates (NHPs), and humans could model in vivo liver-directed AAV expression of valoctocogene roxaparvovec (AAV5-hFVIII-SQ), an experimental gene therapy for hemophilia A with a hepatocyte-selective promoter. Relative levels of DNA and RNA in hepatocytes grown in vitro correlated with in vivo liver transduction across species. Expression in NHP hepatocytes more closely reflected expression in human hepatocytes than in mouse hepatocytes. We used this hepatocyte culture model to assess transduction efficacy of a novel liver-directed AAV capsid across species and identified which of 3 different canine factor VIII vectors produced the most transgene expression. Results were confirmed in vivo. Further, we determined mechanisms mediating inhibition of AAV5-hFVIII-SQ expression by concomitant isotretinoin using primary human hepatocytes. These studies support using in vitro primary hepatocyte models to predict species translatability of liver-directed AAV gene therapy and improve mechanistic understanding of drug-drug interactions.

Ultra-sensitive AAV capsid detection by immunocapture-based qPCR following factor VIII gene transfer.

Adeno-associated virus (AAV)-based gene therapy vectors are replication-incompetent and thus pose minimal risk for horizontal transmission or release into the environment. In studies with AAV5-FVIII-SQ (valoctocogene roxaparvovec), an investigational gene therapy for hemophilia A, residual vector DNA was detectable in blood, secreta, and excreta, but it remained unclear how long structurally intact AAV5 vector capsids were present. Since a comprehensive assessment of vector shedding is required by regulatory agencies, we developed a new method (termed iqPCR) that utilizes capsid-directed immunocapture followed by qPCR amplification of encapsidated DNA. The limit of detection for AAV5 vector capsids was 1.17E+04 and 2.33E+04 vg/mL in plasma and semen, respectively. Acceptable precision, accuracy, selectivity, and specificity were verified; up to 1.00E+09 vg/mL non-encapsidated vector DNA showed no interference. Anti-AAV5 antibody plasma concentrations above 141 ng/mL decreased AAV5 capsid quantification, suggesting that iqPCR mainly detects free capsids and not those complexed with antibodies. In a clinical study, AAV5-FVIII-SQ capsids were found in plasma and semen but became undetectable within nine weeks after dose administration. Hence, iqPCR monitors the presence and shedding kinetics of intact vector capsids following AAV gene therapy and informs the potential risk for horizontal transmission.

Generation of infectious recombinant Adeno-associated virus in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has been successfully employed to establish model systems for a number of viruses. Such model systems are powerful tools to study the virus biology and in particular for the identification and characterization of host factors playing a role in the viral infection cycle. Adeno-associated viruses (AAV) are heavily studied due to their use as gene delivery vectors. AAV relies on other helper viruses for successful replication and on host factors for several aspects of the viral life cycle. However the role of host and helper viral factors is only partially known. Production of recombinant AAV (rAAV) vectors for gene delivery applications depends on knowledge of AAV biology and the limited understanding of host and helper viral factors may be precluding efficient production, particularly in heterologous systems. Model systems in simpler eukaryotes like the yeast S. cerevisiae would be useful tools to identify and study the role of host factors in AAV biology. Here we show that expression of AAV2 viral proteins VP1, VP2, VP3, AAP, Rep78, Rep52 and an ITR-flanked DNA in yeast leads to capsid formation, DNA replication and encapsidation, resulting in formation of infectious particles. Many of the AAV characteristics observed in yeast resemble those in other systems, making it a suitable model system. Future findings in the yeast system could be translatable to other AAV host systems and aid in more efficient production of rAAV vectors.

Selective delipidation of plasma HDL enhances reverse cholesterol transport in vivo.

Uptake of cholesterol from peripheral cells by nascent small HDL circulating in plasma is necessary to prevent atherosclerosis. This process, termed reverse cholesterol transport, produces larger cholesterol-rich HDL that transfers its cholesterol to the liver facilitating excretion. Most HDL in plasma is cholesterol-rich. We demonstrate that treating plasma with a novel selective delipidation procedure converts large to small HDL [HDL-selectively delipidated (HDL-sdl)]. HDL-sdl contains several cholesterol-depleted species resembling small alpha, prebeta-1, and other prebeta forms. Selective delipidation markedly increases efficacy of plasma to stimulate ABCA1-mediated cholesterol transfer from monocytic cells to HDL. Plasma from African Green monkeys underwent selective HDL delipidation. The delipidated plasma was reinfused into five monkeys. Prebeta-1-like HDL had a plasma residence time of 8 +/- 6 h and was converted entirely to large alpha-HDL having residence times of 13-14 h. Small alpha-HDL was converted entirely to large alpha-HDL. These findings suggest that selective HDL delipidation activates reverse cholesterol transport, in vivo and in vitro. Treatment with delipidated plasma tended to reduce diet-induced aortic atherosclerosis in monkeys measured by intravascular ultrasound. These findings link the conversion of small to large HDL, in vivo, to improvement in atherosclerosis.

Delipidated retroviruses as potential autologous therapeutic vaccines--a pilot experiment.

This pilot experiment in a simian immunodeficiency virus (SIV) chronic infection model aimed at extending our previous findings that vaccination with delipidated SIV resulted in more potent and diversified antiviral responses (1). Macaques chronically infected with SIVmac239 treated with antiretroviral therapy (ART) were vaccinated with autologous delipidated virus via consecutive lymph node targeted immunizations-1, 1 and 10 mug of virus spaced monthly. Results showed all animals had lasting viral load reduction approaching 1 log compared to set-point, and disease delay. Delipidation may enhance processing/ presentation of viral antigen eliciting potent antiviral control even at such late infection stage.

Enhancement of cell mediated immune responses using lipid depleted lentivirus as immunogen: a novel approach for inducing recognition of new viral epitopes.

We tested the hypothesis that removal of viral lipids using diisopropylether can enhance antigenicity of SIVmac251. DIPE delipidation removed cholesterol from SIVmac251 without significant loss of viral protein or RNA. Mice immunized with the same SIV preparation but boosted with delipidated SIVmac251 exhibited significantly broader and higher cellular and humoral immune responses compared to live or AT-2-inactivated virus. As little as 1microg (total protein) of delipidated virus was sufficient to induce such enhanced immune responses. Thus, solvent treated lentivirus may provide a novel strategy to generate immune responses to additional viral epitopes.