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Responsible editor | Xi
At present, adeno-associated virus (AAV) has become the main platform for gene therapy delivery in vivo, and AAV has become an important delivery vehicle
for preclinical and clinical research of neurological diseases due to its safe and effective characteristics.
However, gene vector delivery efficiency is an important bottleneck hindering the development of AAV-mediated central nervous system (CNS) gene therapy.
Due to the dense vascular network of the central nervous system, systemic intravenous administration is the best way to achieve widespread distribution of AAV vectors in the brain and spinal cord parenchyma, but adequate trans-blood-brain barrier (BBB) delivery remains a significant obstacle
.
The BBB is made up of cells tightly wedged together that prevent toxins and pathogens that may be present in the blood from entering the brain tissue, while it also blocks potential treatments
for diseases that affect the central nervous system (CNS).
Some natural AAVs have been found to have some ability to cross the blood-brain barrier in mouse models, such as AAV9, AAVrh.
10, and AAVHSC
。 In May 2019, the US FDA approved Zolgensma, the first gene therapy drug using an intravenous AAV9 vector, for the treatment of children under 2 years of age with spinal muscular atrophy caused by the motor neuron survival gene 1 allele, but its price of up to $2.
1 million and the liver damage side effects caused by high doses have also caused more controversy
。 In addition, in order to improve the more efficient AAV that can naturally target the CNS, some AAV variants that can penetrate the blood-brain barrier have been developed with the help of mouse models, including AAV-AS, Anc80L65, AAV.
.
PHP.
B、AAV.
PHP.
eB, AAV-F, and 9P31
.
Despite some progress, it has been relatively difficult
to verify the efficiency of these artificially modified AAVs from rodents to primates 。 For example, AAV.
PHP.
B has a very good infection efficiency on the central nervous system in the C57BL/6 mouse strain after intravenous administration, but American scholar James Wilson and Japanese scholar Hirokazu Hirai respectively reported that AAV.
PHP.
B did not show better blood-brain barrier penetration efficiency
than AAV9 in BALB/cJ strain mice and non-human primates (NHP).
On October 10, 2022, the Fengfeng Bei team of the Department of Neurosurgery at Brigham and Women's Hospital at Harvard University in the United States published a paper entitled " Research article on Variants of the adeno-associated virus serotype 9 with enhanced penetration of the blood–brain barrier in rodents and primates.
In this study, the mutant library was obtained by modifying the AAV9 viral capsid by rational design, and AAV.
CPP.
16 with the characteristic of penetrating the blood-brain barrier was successfully screened, and verified on animal models of different strains of mice and cynomolgus monkeys, and it was found that intravenous injection of AAV.
CPP.
16 could effectively effect the blood-brain barrier and infect various cells
of the central nervous system brain and spinal cord 。 Subsequently, it was found that the use of AAV.
CPP.
16 as a delivery vehicle also had a good antitumor effect
on mouse models of malignant glioma.
First, the authors inserted cell-penetrating peptide (CPP) at the VR-VIII capsid bulge 588/589 site of AAV9 based on rational design The mutant library was obtained and packaged to produce AAV, and then each mutant AAV was injected into the hybrid mice of C57BL/6J and BALB/cJ lines by tail vein injection for screening, and AAV.
CPP.
11 and AAV.
CPP.
12
with better blood-brain barrier penetration than AAV9 were obtained 。 Subsequently, the 5-mer CPP.
11 and CPP.
12 peptides from the Ku70 family Bip1 and Bip2 peptides were optimized and expanded for further screening and verification, and the 6-mer CPP.
16 and 7-mer CPP.
21
that could effectively cross the blood-brain barrier in hybrid mice were successfully obtained.
In the validation of different mouse strains, the results showed that AAV.
CPP.
16 and AAV.
CPP.
21 could increase the efficiency
of brain infection by about 6-249 times compared with AAV.
21.
In order to further verify the infection efficiency of the new AAV on primates, the researchers tested two new AAV and AAV9 on adult cynomolgus monkeys and neonatal cynomolgus monkey models, respectively, and the efficiency of CPP.
16 penetrating the blood-brain barrier was about 5 times higher than that of AAV9 on average
.
To further investigate the affinity of AAV.
CPP.
16 for infection of different cells of the central nervous system, the authors found that intravenous administration of 1e+12 vg AAV to BALB/cJ mice effectively infected about 10-51% of brain neuronal cells, 20-49% of astrocytes, and 64-72% of spinal cord motor neurons
。 More importantly, analysis of cynomolgus monkey samples found that AAV.
CPP.
16 still exhibits high neuronal affinity for astrocytes and is able to mediate infestation of about 25% of spinal motor neurons
.
Based on the results of mouse and cynomolgus monkeys, intravenous AAV can still infect peripheral organs, such as liver, muscle, heart and dorsal root ganglia, but does not cause significant sustained hepatotoxicity and dorsal root ganglia degeneration
.
For the mechanism study of AAV.
CPP.
16, the authors first explored the impact on the integrity of the blood-brain barrier and found that the characteristics of AAV.
CPP.
16 across the blood-brain barrier did not cause the permeability of the blood-brain barrier to be damaged
.
Using the in vitro BBB model test developed by Choi-Fong, it was proved that AAV.
CPP.
16 can significantly improve the transcytosis
of epithelial cells.
In vitro cell experiments also showed that AAV.
CPP.
16 significantly increased the infection efficiency of different cells and the binding efficiency
of epithelial cells and HEK293T cells compared with AAV9.
Subsequently, the authors used the mouse malignant glioma GL261 model to explore the effect
of AAV.
CPP.
16 as a gene therapy vehicle for in vivo delivery of secretion-targeted PD-L1 antibody drugs and the non-secretory suicide gene HSV-TK1 。 The results of PD-L1 antibody delivery based on AAV.
CPP.
16 showed that AAV.
CPP.
16 in the malignant glioma microenvironment was about 17.
5 times more effective than AAV9-delivered antibody, significantly increased CD8+T infiltration and IFNg secretion, and reduced Treg cell infiltration, thereby effectively improving the survival rate of tumor-bearing mice to 75%, and the survival rate of recurrent tumor models in long-term surviving mice reached 100%.
Using the HSV-TK1 suicide gene system as a therapeutic tool, the researchers added the miR122 target sequence to the AAV vector to inhibit the toxic cell damage effect of
peripheral tissues.
AAV.
CPP.
16-mediated HSV-TK1 therapy combined with intraperitoneal injection of GCV can significantly kill tumor cells, reduce tumor volume, and improve the survival rate of mice to about 57%.
In summary, this study found that AAV.
CPP.
16 can effectively pass through the blood-brain barrier of the central nervous system and deliver drugs to the brain and spinal cord through screening, and fully demonstrated the therapeutic efficacy
of the vector in mouse malignant glioma models.
Professor Fengfeng Bei, corresponding author of the paper, said: "Our study is gratifying because we are one step
closer to successfully translating AAV-delivered drugs across the human blood-brain barrier.
Our study also suggests that AAV can be used as an effective systemic drug delivery tool to fight gliomas or other in-demand central nervous system diseases
.
It is reported that Fengfeng Bei has applied for a number of related patents and established the AAV gene therapy startup Brave Bio Inc.
Original link:
https://doi.
org/10.
1038/s41551-022-00938-7
Pattern maker: Eleven
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