The presence of CpGs in AAV gene therapy vectors induces a plasmacytoid dendritic cell-like population very early after administration.

AAV-mediated gene transfer is a promising platform still plagued by potential host-derived, antagonistic immune responses to therapeutic components. CpG-mediated TLR9 stimulation activates innate immune cells and leads to cognate T cell activation and suppression of transgene expression. Here, we demonstrate that CpG depletion increased expression of an antibody transgene product by 2-3-fold as early as 24 h post-vector administration in mice. No significant differences were noted in anti-transgene product/ anti-AAV capsid antibody production or cytotoxic gene induction. Instead, CpG depletion significantly reduced the presence of a pDC-like myeloid cell population, which was able to directly bind the antibody transgene product via Fc-FcγR interactions. Thus, we extend the mechanisms of TLR9-mediated antagonism of transgene expression in AAV gene therapy to include the actions of a previously unreported pDC-like cell population.

Kinetics and durability of transgene expression after intrastriatal injection of AAV9 vectors.

Understanding the kinetics and durability of AAV-mediated transgene expression in the brain is essential for conducting basic neuroscience studies as well as for developing gene therapy approaches for CNS diseases. Here, we characterize and compare the temporal profile of transgene expression after bilateral injections into the mouse striatum of rAAV9 encoding GFP under the control of either a ubiquitous promoter (CAG), or the neuron-specific human synapsin (hSyn) and CamKII promoters. GFP protein expression with the CAG promoter was highest at 3 weeks, and then decreased to stable levels at 3 and 6 months. Surprisingly, GFP mRNA levels continued to increase from 3 weeks to 3 months, despite GFP protein expression decreasing during this time. GFP protein expression with hSyn increased more slowly, reaching a maximum at 3 months, which was equivalent to protein expression levels from CAG at that time point. Importantly, transgene expression driven by the hSyn promoter at 6 months was not silenced as previously reported, and GFP mRNA was continuing to rise even at the final 6-month time point. Thus, hSyn as a promoter for transgene expression demonstrates long-term durability but may require more time after vector administration to achieve steady-state levels. Because CAG had the highest GFP protein expression in our comparison, which was at 3 weeks post administration, the early kinetics of transgene expression from CAG was examined (1, 2, 5, and 10 days after injection). This analysis showed that GFP protein expression and GFP mRNA increased during the first 3 weeks after administration. Interestingly, vector DNA rapidly decreased 10-fold over the first 3 weeks following injection as it assembled into stable circular episomes and concatemers. Surprisingly, the processing of vector genomes into circular episomes and concatemers was continually dynamic up to 3 months after injection. These results provide novel insight into the dynamic processing of vector genomes and promoter-specific temporal patterns of transgene expression in the brain.

Directed Evolution of AAV Serotype 5 for Increased Hepatocyte Transduction and Retained Low Humoral Seroreactivity.

Most recombinant adeno-associated virus (AAV) capsids utilized in liver gene therapy have significant levels of pre-existing neutralizing antibodies in the human population. These neutralizing factors limit the patient pools eligible for receiving AAV-mediated therapies. AAV serotype 5 (AAV5) does not face the same barrier of humoral immunity as most AAV serotypes due to its low seroprevalence. However, AAV5 can only facilitate a low level of transgene expression in the liver, constraining its application to a small number of liver diseases. To improve the liver transduction of AAV5 while retaining its low seroprevalence, we constructed a library of AAV5 mutants via random mutagenesis and screened in Huh7 cells. Two molecularly evolved AAV5 variants, MV50 and MV53, demonstrated significantly increased transduction efficiency in Huh7 cells (∼12×) and primary human hepatocytes (∼10×). All variants had retained low seroreactivity toward pooled human immunoglobulin G (IgG) when compared to AAV5, which was significantly less seroreactive than AAV9. Functional characterization of the mutants also revealed insights into the functions of various domains, especially the VR-I, in the AAV5 capsid. The result is AAV5 variant capsids with much enhanced human hepatocyte transduction, potentially useful for liver-directed gene therapy.