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Adeno-associated viruses (AAV) are the main means of delivering therapeutic gene cargo to target cells for gene therapy. However, natural AAVs are not specifically targeted at diseased cells and tissues, in addition, they can be recognized and destroyed by the immune system – this limits the effectiveness of therapy. To improve the work of AAV, biologists used the method of "directed evolution", in which mutations are randomly produced in the building blocks of capsid proteins that form the envelope of the virus and bind directly to target cells. By evaluating which changes help target the virus to target tissues, and sequentially accumulating mutations one after another, researchers are trying to improve the necessary characteristics of AAV.
In this study, scientists from the Harvard Institute of Biological Engineering and Harvard Medical School have created a method to accelerate the process of creating improved outer shells (capsids) of AAV and develop a better version of viruses for gene therapy.
Proteins of the capsid of the adenoassociated virus of the most studied serotype AAV-2.
The researchers mutated sequentially in each of the 735 amino acids in the AAV2 capsid, including all possible codon substitutions, insertions and deletions at each position. Thus, a virus library containing about 200,000 mutation variants was created. The researchers identified changes in the capsid that simultaneously maintained the viability of AAV2 and increased its tropicity ("force of attraction") to specific organs in mice.
To create one of the most complete AAV capsid libraries to date, a group of researchers led by George Church and Eric Kelsick used the entire arsenal of tools and methods of synthetic biology, including the possibilities of synthesis, coding and sequencing of a new generation of DNA. With the help of the information contained in this library, they were also able to develop capsids with a number of mutations exceeding the previous natural or synthetic variants. The efficiency of viable capsids far exceeds that of AAVs created by random mutagenesis methods.
The resulting library of mutations of genes encoding the AAV2 capsid was subjected to a broad phenotypic analysis, including the production of viruses, the immune response of the host organism to them, thermal stability and bio-distribution. The distribution of the created AAV mutants by major organs in mice revealed dominant trends affecting in vivo delivery.
By chance, an auxiliary protein was discovered hidden in the DNA sequence encoding a capsid that binds to the membrane of target cells. This membrane-bound auxiliary protein (MAAP) exists in all the most popular AAV serotypes and is involved in the natural life cycle of the virus. The authors believe that understanding the functions of MAAP can give rise to future research and lead to improvements in gene therapy using AAV.
Article by P.J.Ogden et al. Comprehensive AAV capsid fitness landscape reveals a viral gene and enables machine-guided design published in the journal Science.
Aminat Adzhieva, portal "Eternal Youth" http://vechnayamolodost.ru based on the materials of the Wyss Institute: Wyss Institute researchers demonstrate machine-guided engineering of AAV caps for gene therapy.