New viral vectors for gene delivery successfully penetrate the brain of macaques
Tatiana Sashina, PCR.news
Gene therapy has enormous potential, but the targeted delivery of functioning copies of genes to those tissues and organs where they are needed is an extremely difficult task. Vectors based on the adenoassociated virus (AAV) appear to be a promising tool for delivering genes to the central nervous system after intravenous infusion. Clinical and preclinical studies of recombinant AAV (rAAV) with natural AAV serotypes have shown promising results in the treatment of CNS disorders, including Alzheimer's disease, Parkinson's disease, spinal muscular atrophy. However, natural variants of the virus hardly penetrate the blood-brain barrier (BBB) and accumulate in the liver. Therefore, it is necessary to increase the doses of AAV so that at least a small part of them gets into the target tissue. In some cases, such high doses led to liver damage. In addition, the transition from testing vectors on model mice to experiments on monkeys turned out to be very difficult.
Researchers from the Fords Institute of the Massachusetts Institute of Technology and Harvard have developed a family of AAVs capable of penetrating such a complex target tissue as the brain. They are more than three times better at delivering their important cargo to the primate brain than AAV9, the only viral vector penetrating the BBB and approved by the FDA for the treatment of CNS diseases.
To detect AAVs that overcome the blood-brain barrier, the scientists used a directed evolution strategy implemented on the basis of the DELIVER technology they had previously developed. They constructed a huge pool of randomly generated capsids based on AAV9. A random peptide of 7 amino acid residues was inserted into the hypervariable region of the VR-VIII capsid protein between the residues Q588 and A589 — just in the place that is located on the surface of the capsid. Each variant of the capsid packed its own coding sequence, which was achieved by flanking the transgene with inverted terminal repeats (ITR). In order to select those capsid variants that predominantly transduce neurons, scientists placed the transgen under the control of a human neuron-specific synapsin promoter.
Next, the researchers narrowed the number of capsid variants obtained to those that are able to penetrate the brains of mice and macaques. After two rounds of in vivo breeding on 48 C57BL/6J and BALB/cJ mice, as well as on five young Javanese macaques, scientists identified a family of capsids designated PAL. When evaluating the effectiveness of several constructed raavs on macaques, three variants of the first-generation PAL (PAL1A-C) capsid showed a two- to three-fold increase in the expression of transgene mRNA throughout the brain. In an experiment with one macaque, the second-generation PAL2 variant demonstrated an even greater (4-6 times) increase in the expression of transgene mRNA in most areas of the brain. PAL2 was also 13 times more active than AAV9 in neural mesh transduction. Perhaps the systemic administration of rAAV will allow genes to be delivered not only to the brain, but also to the retina, for example, for the treatment of Crabbe disease.
In addition to increased tropism to the central nervous system in macaques, PAL variants of the first generation (PAL1A-C) demonstrated a significant decrease in tropism to the kidneys, lungs, thymus and liver of macaques. Both the delivery of the vector genome and the expression of the transgene mRNA decreased. The second generation vector PAL2 also did not accumulate in the liver.
Scientists have found that variants designed for mice did not always work successfully in the central nervous system of macaques. In this work, four mouse vectors were selected using DELIVER, which effectively transduced the CNS of two mouse lines. However, none of them surpassed AAV9 in transduction of any area of the macaque brain.
Thus, the PAL family is a class of AAV—based vectors designed for primates that overcome the blood-brain barrier more effectively than AAV9, have reduced tropism to the liver and have great therapeutic potential. The results of the work also emphasize the urgent need to use appropriate animal models to search for and evaluate new AAVS designed to deliver genes to the human central nervous system. Such a delivery system can be used for the treatment of intractable neurological diseases.
Article by Stanton et al. Systematic administration of novel engineered AAV capsids facilitates enhanced transgene expression in the macaque CNS is published in the journal Med.
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