Virus against glioblastoma
AAV9-based vector penetrates into the brains of mice and primates with intravenous administration
Irina Konova, PCR.news
Gene therapy vectors based on adenoassociated viruses (AAV), aimed at combating diseases of the central nervous system, would be most convenient to introduce systemically, since the spinal cord and brain are rich in blood vessels. However, a serious obstacle stands in the way of vectors — the blood-brain barrier. Some AAVs, for example, AAV9, have a natural ability to overcome the BBB.
Scientists use various approaches to improve the efficiency of vector delivery to the central nervous system, but not always the results achieved in mice are reproduced in primates. In the new work, a team from the USA optimized AAV9 using cell-penetrating peptides (CPP). The obtained vector variants were tested on mice and crab-eating macaques.
CPP is a group of short peptides capable of crossing biological membranes and facilitating the transport into the cell of molecules that otherwise cannot get into it. Scientists have suggested that by placing certain CPPS on the surface of the AAV9 capsid, it is possible to modulate the tissue tropism of the vector. They tested 14 CPP. The genetic sequence of peptides was embedded in the sequence of the receptor-binding region of the capsid. In addition, a red fluorescent protein (RFP) gene was placed on the vector, which was used as a reporter.
Nine of the 14 AAV variants did not produce sufficient viral titers in vitro. The ability of the remaining five to cross the BBB was tested on mice. The original AAV9 modified by RFP was used as a control. Mice were injected intravenously with one of the AAV variants, and their brains were examined three weeks later. Two vectors, AAV.CPP.11 and AAV.CPP.12, demonstrated an increased ability to pass through the BBB compared to AAV9. They carried CPP Bip1 and Bip2, respectively. These molecules belong to the family of 5-dimensional Bax-inhibiting peptides derived from the Ku70 protein. Scientists modified the sequences of these peptides by adding and rearranging amino acids and thus increasing the tropism of the vector to brain cells. The final variants, AAV.CPP.16 and AAV.CPP.21, passed through the BBB of mice of various lines with intravenous administration tens and hundreds of times more effectively than AAV9. At the same time, as shown by experiments on cells, the vectors did not suppress the expression of the pro-apoptotic Bax gene, despite the origin of CPP.
At the next stage, the scientists tested AAV.CPP.16 and AAV.CPP.21 on young and adult crab-eating macaques. The experiment involved nine monkeys with a previously confirmed absence of neutralizing antibodies to AAV9. Instead of fluorescent protein, human aromatic amino acid decarboxylase (hAADC) was used as a reporter, since it does not cause an immune response in lower primates. The reporter in the brain cells was visualized using immuno-staining. AAV.CPP.16 showed the best results. It passed through the BBB about five times better than the original AAV9, and worked equally well in young and adult macaques.
Then the scientists returned to the mice. In a mouse model of glioblastoma, they showed that AAV.CPP.16, when administered intravenously, can carry a therapeutic load through the BBB — a sequence of a variable fragment of an antibody to mouse PD-L1. Mice were inoculated with tumor cells and a vector was injected five days later. Using an immunoassay conducted on the 19th day of the experiment, the scientists confirmed the expression of the therapeutic molecule in tumor cells. In addition, they observed the immunomodulatory effect of PD-L1 blockade and increased survival of mice that received the therapeutic vector.
Thus, the authors obtained a vector based on AAV9, which effectively penetrates into the brains of mice and primates. They plan to improve it further, in particular, to more strictly limit its tropism (in an experiment on a glioblastoma model, a therapeutic molecule was also found in the liver and muscles of mice).
Article by Yao et al. Variants of the adeno-associated virus serotype 9 with enhanced penetration of the blood–brain barrier in rodents and primates is published in the journal Nature Biomedical Engineering.
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