A Chinese study led by scientists at Nanjing University is the first to show that modified small extracellular vesicles (sEVs) can cross the placenta and blood-brain barrier (BBB) to target drug delivery in mice infected with the Zika virus (ZIKV), in which they were shown to significantly reduce fetal neurological damage, including microcephaly.

"Targeted delivery via modified sEVs may be a promising alternative to traditional delivery methods, especially for treating brain viral infections," said study leader Zhiwei Wu, a professor and director of the Center for Public Health Research at Nanjing University Medical School.

"Increasing the yield and efficiency of producing sEVs and developing those that can target other tissues will broaden their application and expand the effectiveness of this gene delivery technique."

ZIKV represents a significant global health threat, resulting in miscarriages or causing neurological and congenital neonatal sequelae, most notably microcephaly and the autoimmune disorder, Guillain-Barre syndrome.

This is because ZIKV can cross both the placenta and the BBB, which effectively prevents large molecules, including most drugs, from entering the brain.

"There are few specific drugs or transporters targeting brain tissue, and most of those are highly toxic and are not efficient in crossing the BBB," noted Wu.

Currently, no ZIKV-specific therapies or preventive vaccines are available, meaning that there is an urgent need for safe and effective antivirals that can effectively cross the placenta and BBB, particularly those that prevent microcephaly.

Gene-silencing immunotherapies using oligonucleotides are particularly promising candidates in this respect, having shown unique advantages in clinical settings, but their delivery into cells remains a major challenge.

"The delivery of oligonucleotides to treat diseases is challenging because of their short half-life, limited cell uptake, and poor tissue penetration," said Wu.

This has sparked interest in the use of alternative strategies for oligonucleotide delivery, for which modified sEVs seem particularly promising.

Modified sEVs

Endogenous biodegradable nanoparticles, cellular sEVs are important mediators of intracellular communication, which evidence increasingly suggests could be powerful and potentially safe tools for delivering encapsulated treatments.

Moreover, "nanomolecules are being designed as delivery systems for the treatment of neurological diseases, including Alzheimer's and Parkinson's disease, brain tumors, and stroke," noted Wu.

"Cellular sEVs are emerging as a powerful tool for the treatment of various diseases, including cancer, and cardiovascular and infectious diseases, with engineered sEVs being increasingly used in tumor therapies, immunotherapies, and antiviral research," he said.

For example, Wu's team at Nanjing University has previously reported having modified sEVs to deliver an antiviral across the placenta to inhibit ZIKV infection in fetal mice.

"Our previous study indicated that sEVs were able to deliver a placenta-bound antiviral molecule across the placental barrier to inhibit ZIKV infection of mouse fetuses," he said.

In their latest study reported in the November 10, 2021, edition of Molecular Therapy, Wu and his team showed that sEVs could deliver antiviral treatments to target ZIKV in the fetal brain and prevent neurological damage.

They genetically engineered sEVs expressing neuron-specific rabies virus glycoprotein (RVG) on their surface in order to target neurons.

The researchers then loaded the modified sEVS with small interfering RNA (siRNA) to specifically inhibit ZIKV replication and protein transcription, and injected them into pregnant mice.

The RVG-modified sEVs were demonstrated to cross both the placenta and the BBB, protecting against ZIKV transmission to the fetus.

Notably, in the fetal brain, the modified sEVs were demonstrated to significantly suppress ZIKV infection, as demonstrated by assessing the post-mortem viral load versus non-infected controls.

Importantly, compared with untreated controls, the treatment was also demonstrated to reduce inflammation and neurological damage, including microcephaly and cerebral defects, in postmortem pups.

Together, these findings indicate that modified sEVs containing siRNA "can provide an ideal method to enhance delivery of cargo such as nucleic acids, achieving targeted treatment of brain viral infection and control of related neurological damage," said Wu.

However, many unanswered questions remain about this promising treatment strategy. For example, the viral vector and the first treatment dose were administered simultaneously, so it remains unclear whether later treatment would also be effective.

"A delayed injection after viral infection may provide more confidence in the ability to translate this research to human trials," said Wu. "Nevertheless, our study provides a proof of concept for such a possibility."

In the future, the researchers are planning to investigate the molecular mechanisms whereby sEVs penetrate the placenta and BBB, while determining those factors that control their effective delivery.

"Since sEVs are of biological origin, they can be a safe drug delivery vehicle, which overcomes the limitations of clinical gene therapy, including poor targeting, lack of effective gene therapeutic tools, a short serum half-life, and high immunogenicity," said Wu.

However, he cautioned, "the current study is preliminary and many more issues need to be resolved before the immunotherapy could be considered for human use."