Yubo Liu,Fangming Liang,Rui Zhang,Chengcheng Tang,Xin Li,Yongjun Wang,Zhonggui He,Hongzhuo Liu
Recombinant adeno-associated virus (AAV) vectors have become the promising platform to deliver a therapeutic transgene for the treatment of monogenic disorders. However, anti-AAV antibodies that are released by B cells after AAV treatment create a substantial barrier to their long-lasting safe and effective therapy, making AAV gene therapy a “one shot” treatment. Here, we offered that tolerogenic nano-adjuvants (named RICP) composed of rapamycin (RAPA) and itaconate (ITA), to induce specific immune tolerance to overcome the anti-drug antibodies (ADAs). Such strategy blocked the feedback loops of follicular helper T （Tfh） cells and germinal center B cells by metabolic regulation, complementary to induction of regulatory T cells (Tregs) by mTORc1 inhibitor RAPA, consequently abolished the secretion of anti-AAV antibodies. The developed nano-adjuvants can also significantly increase expression of a transgene (such as luciferase, green fluorescent protein and human embryonic alkaline phosphatase reporter transgene) in the liver at re-dosing, compared to mice treated with AAV alone which showed almost no change in transgene expression. The emerging strategy provided AAV re-administration schedule available for not only mitigate toxicities with high vector doses, but re-storing therapeutic benefits in children in significant cell proliferation.
To overcome limitations in generalizability and efficiency of current AAV vectors, in this current study, we constructed an AAV variant library by insertion of random heptapeptide sequences in the receptor-binding domain of the AAV9 capsid gene. We then applied a recently developed organ-on-a-chip in vitro model of human blood-brain barrier (BBB) to iteratively enrich for variants that efficiently cross the BBB and transduce astrocyte cells. Through multiple rounds of screening, we obtained two candidate AAV variants, AAV-M6 and AAV-M8, that showed significantly higher BBB penetration efficiency than AAV9 or AAV-PHP.eB. Quantitative PCR (qPCR) assay showed that AAV-M6 could accumulate to 5 times higher titer, while AAV-M8 reached 3 times higher titer, than AAV-PHP.eB in the neural chamber of the model. Transduction assay further verified that the AAV-M6 candidate vector was able to infect HA-1800 cells after crossing the BBB, suggesting it could potentially transduce brain parenchymal cells after crossing the hCMEC/D3 layer at higher efficiency than AAV-PHP.eB. Molecular simulations suggested that the human receptor proteins, LY6D and M6PR, could bind the AAV-M6 heptapeptide insertion with high affinity. This study provides two promising candidate AAV vectors and demonstrates the use of this in vitro BBB model for scalable, high throughput screening of gene therapies. These tools can drive investigations of the mechanisms underlying BBB permeability and the cell type specificity of virus vectors.
Prion diseases are fatal neurodegenerative disorders affecting humans and animals, and the central pathogenic event is the conversion of normal prion protein (PrPC) into the pathogenic PrPScisoform. Previous studies have identified nanobodies that specifically recognize PrPCand inhibit the PrPCto PrPScconversion in vitro. In this study, we investigated the potential for in vivo expression of anti-PrPCnanobodies and evaluated their impact on prion disease. The coding sequences of three nanobodies were packaged into recombinant adeno-associated virus (rAAV) and were administered via intracerebroventricular (ICV) injection in newborn mice. We found that the expression of these nanobodies remained robust for over 180 days, with no observed detrimental effects. To assess their therapeutic potential, we performed ICV injections of nanobody-expressing rAAVs in newborn mice, followed by intracerebral prion inoculation at 5–6 weeks of age. One nanobody exhibited a small yet statistically significant therapeutic effect, extending survival time from 176 days to 184 days. Analyses of diseased brains revealed that the nanobodies did not alter the pathological changes. Our findings suggest that high levels of anti-PrPCnanobodies are necessary to delay disease progression. Further optimization of the nanobodies, AAV vectors, or delivery methods is essential to achieve a significant therapeutic effect.
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